Effect of divalent metal cations on hydroxyapatite ... · resembling dental caries, or 0.3% citric...
Transcript of Effect of divalent metal cations on hydroxyapatite ... · resembling dental caries, or 0.3% citric...
Effect of divalent metal cations on hydroxyapatite dissolution kinetics
relevant to dental caries and erosion.Lingawi, Hanadi Saud
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EFFECT OF DIVALENT METAL CATIONS
ON HYDROXYAPATITE DISSOLUTION
KINETICS RELEVANT TO DENTAL
CARIES AND EROSION
HANADI SAUD LINGAWI
BDS MSc Dent Rad MClin Dent Paeds
Thesis submitted in fulfilment of the requirements for the degree of Doctor of
Philosophy in the Faculty of Medicine, University of London
May 2012
Centre for Oral Growth and Development
Institute of Dentistry
Queen Mary’s School of Medicine and Dentistry
University of London
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Declaration regarding plagiarism
I declare that the coursework material attached herewith is entirely my own work
and that I have attributed any brief quotations both at the appropriate point in the text
and in the bibliography at the end of this piece of work.
I also declare that I have not used extensive quotations or close paraphrasing and that
I have neither copied from the work of another person, nor used the ideas of another
person, without proper acknowledgement.
Name: Hanadi Saud Lingawi Course: PhD
Title of work submitted:
Effect of Divalent Metal Cations on Hydroxyapatite Dissolution Kinetics Relevant to
Dental Caries and Erosion
Examination: A thesis submitted for the degree of Doctor of Philosophy, University
of London
Signature: Date
ABSTRACT
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Abstract
In recent years there has been an increasing awareness of the influence of
various trace elements on reducing the progression of dental caries and of erosion.
However, there are few clinical and even fewer in-vitro studies of the cariostatic
effect of some trace elements on the progression of dental caries. Further, there is
currently no consensus on the underlying physico-chemical mechanism on the
influence of trace elements on these processes.
The aim of this study was to investigate the effect of three divalent cations;
zinc (Zn2+
), strontium (Sr2+
) and copper (Cu2+
), on the physical-chemistry
influencing hydroxyapatite (HAp) dissolution kinetics, using scanning
microradiography (SMR), under simulated cariogenic and erosive conditions
relevant to the oral environment.
Compressed and sintered porous HAp discs were used as model systems for
dental enamel. These discs were exposed to demineralising solutions containing a
range of concentrations of Zn2+
, Sr2+
and Cu2+
, and either 0.1% acetic acid at pH 4.0
resembling dental caries, or 0.3% citric acid at pH 2.8 resembling erosion conditions.
SMR is a development of the photographic microradiography technique of
mineral quantification by means of X-ray absorption, but allows real-time
quantification measurement of the rate of HAp mineral loss (RDHAp). Sequential
SMR experiments during which the HAp disc was exposed to demineralising
solution, containing each cation in either increasing or decreasing concentration
order (separated by 30 minutes of washing with de-ionised water) allowed evaluation
of the persistence of the influence of the divalent cations being investigated.
The results showed that all three divalent cations decreased RDHAp
significantly under both investigated conditions but via two different mechanisms.
It was proposed that Zn2+
and Cu2+
decrease the RDHAp through a surface
controlled mechanism whereas Sr2+
decreases the RDHAp through a solid phase
change. This information will be useful as part of the development of therapeutic
products which include these ions for the prevention of dental caries and erosion.
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I dedicate this research work to the soul of my beloved mother
Hayat Bakhsh
(1941-2012)
May God rest her soul in Heaven
ACKNOWLEDGEMENTS
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Acknowledgements
Here I would like to thank all those who were involved in and supported me in my
PhD research.
I am thankful to my academic supervisor, Dr Paul Anderson, who stood by me
through this entire PhD journey. Also I would like to thank my second supervisor, Dr
Michele Barbour from the University of Bristol, for making me feel welcome, giving
me access to her department facilities and giving me the chance to experience and
enjoy the taste of collaborative work between different institutes.
I am also grateful to Dr Richard Lynch from GlaxoSmithKline (GSK) and Honorary
Research Fellow at University of Liverpool, who was generous with his time and
advice regarding our zinc experiments; Dr Rory Wilson for his help with XRD;
Professor Robert Hill for his enriching discussions linking the academic research and
industrial worlds; Dr Natalia Karpukhina for her valuable discussions about
strontium; and Dr Siân Jones from the University of Bristol for her patient tutoring
that made my trips to Bristol such a joy.
A special thanks to Dr Sharif Islam at QMUL who offered me the guidance during
the statistical analysis of the data.
I cannot thank enough Professor Mark Hector, now the Dean of Dentistry at
University of Dundee, who has been a great support and enormous help during the
process of my GDC registration and during my work as an honorary clinical lecturer
at the Paediatric Dentistry Department at QMUL.
I am deeply thankful to Dr Jacqueline Brown at King’s College, University of
London for being such an inspiration since I was her student at King’s College
during my MSc in Dental Radiology course.
ACKNOWLEDGEMENTS
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I wish to thank the Dental Materials Science Laboratory at the School of Oral and
Dental Sciences, University of Bristol for supplying this project with the HIMED
hydroxyapatite discs.
My deep appreciation and gratitude go to the Saudi Ministry of Higher Education
and the Saudi Cultural Bureau in UK for their financial grant and their continuous
support throughout my course of studies.
My immense gratitude goes to my parents for their continued love and support, and
to my sister Dr Arij, without whose encouragement I would have never reached this
stage of my PhD.
TABLE OF CONTENTS
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Table of contents
Declaration 2
Abstract 3
Dedication 4
Acknowledgements 5
Table of contents 6
List of figures 15
List of tables 22
List of abbreviations 24
PART I: INTRODUCTION AND LITERATURE REVIEW
CHAPTER 1: Introduction
1.1 General introduction 26
1.2 General aim 28
1.3 Thesis layout 28
CHAPTER 2: Human Dental Enamel
2.1 Dental enamel chemical composition 30
2.2 Dental enamel structure 31
2.3 Physical properties of dental enamel 32
2.4 Trace elements in dental enamel 33
2.4.1 Carbonate 34
2.4.2 Fluoride 35
2.4.3 Magnesium 35
2.5 Hydroxyapatite as a model system for dental enamel 36
CHAPTER 3: Dental Enamel Caries and Erosion
3.1 Dental enamel caries 39
3.1.1 Introduction to dental enamel caries 39
3.1.2 Aetiology of dental caries 40
TABLE OF CONTENTS
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3.1.3 Histology and chemical changes in enamel caries 41
3.1.4 Methods of dental caries detection 44
3.1.5 Prevalence of dental caries 45
3.2 Dental erosion 48
3.2.1 Introduction to dental erosion 48
3.2.2 Aetiology of dental erosion 49
3.2.3 Prevalence of dental erosion 52
3.2.4 Methods of dental erosion detection and assessments 53
3.3 Laboratory techniques for assessment of dental hard tissue loss 54
3.3.1 Scanning electron microscopy 54
3.3.2 Environmental scanning electron microscopy 55
3.3.3 Atomic force microscopy 55
3.3.4 Surface profilometry 55
3.3.5 Nanoindintation and microindintation 56
3.3.6 Chemical analysis 56
3.3.7 Microradiography 57
CHAPTER 4: Calcium Apatite Dissolution Models
4.1 Introduction 58
4.1.1 Diffusion controlled and surface controlled models 58
4.1.2 Self inhibition (calcium rich layer formation) model 59
4.1.3 Stoichiometric/Non-stoichiometric dissolution model 60
4.1.4 Chemical model 60
4.1.5 Nanoscale enamel dissolution model 61
4.2 Summary 62
CHAPTER 5: Zinc
5.1 Introduction 63
5.2 Zinc in the oral cavity 65
5.3 Effect of zinc on calculus formation 66
5.3.1 Zinc containing mouthwashes 66
5.3.2 Zinc containing toothpastes 67
5.4 Effect of zinc on dental caries 69
TABLE OF CONTENTS
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5.5 Effect of zinc on dental erosion 70
5.6 Effect of zinc on hydroxyapatite dissolution 70
CHAPTER 6: Strontium
6.1 Introduction 73
6.2 Strontium in bone 75
6.3 Strontium in the oral cavity 76
6.4 Effect of strontium on hydroxyapatite dissolution 77
6.5 Effect of strontium on dental caries 77
6.6 Effect of strontium on dentine hypersensitivity 79
CHAPTER 7: Copper
7.1 Introduction 81
7.2 Effect of copper on dental plaque 82
7.3 Effect of copper on dental caries 84
7.4 Effect of copper on enamel demineralisation 85
CHAPTER 8: X-ray Microscopy
8.1 Nature of electromagnetic radiation 88
8.2 X-ray generation 89
8.2.1 Introduction 89
8.2.2 Modern X-ray tube 90
8.2.3 Microfocus tubes 93
8.2.4 Electron impact X-ray source 93
8.2.5 Factors affecting X-ray beam quantity and quality 94
8.3 X-ray interaction with matter 97
8.3.1 Attenuation mechanisms 97
8.3.2 X-ray attenuation Beer’s law 99
8.3.3 Types of attenuation coefficient (LAC) 100
8.4 X-ray detection 100
8.4.1 Introduction to semiconductors 100
8.4.2 Multichannel analysers (MCA) 101
TABLE OF CONTENTS
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CHAPTER 9: Scanning Microradiography Theory and Methodology
9.1 Introduction 102
9.2 SMR system apparatus 104
9.2.1 X-ray generator 105
9.2.2 X-ray detector 105
9.2.3 SMR stage 105
9.2.4 SMR cells 106
9.2.5 Area scanning 106
9.2.6 Data analysis 107
PART II: METHODOLOGY
CHAPTER 10: Modification of Real-Time Scanning Microradiography for
The Quantitative Measurements of Dissolution Kinetics of Compressed
Permeable Hydroxyapatite Discs Over Short Period of Time
10.1 Introduction 111
10.2 SMR system apparatus used in this study 112
10.2.1 X-ray generation 112
10.2.2 X-ray detector 113
10.2.3 SMR stage 114
10.3 Area scanning 115
10.4 Data analysis at a point 115
10.5 The effect of SMR data sampling frequency on the statistics of
mineral mass loss calculation
116
10.5.1 Effect of even sampling frequency
10.5.2 Effect of multiple SMR cells simultaneous scanning
117
121
10.6 SMR cell design and specimen preparation 124
10.6.1 SMR cells 124
10.6 2 Specimen preparation 126
10.7 Demineralisation solutions 127
10.7.1 0.1% acetic acid pH 4.0 127
10.7.2 0.3% citric acid pH 2.8 128
TABLE OF CONTENTS
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PART III : DEVELOPMENT OF A PROTOCOL
Introduction to Development of a Protocol
CHAPTER 11: Characterisation of HIMED and Plasma-Biotal Compressed
Hydroxyapatite Disc
130
11.1 Introduction 132
11.2 Aims and objectives 132
11.3 Materials and methods 133
11.3.1 X-ray microtomography 133
11.3.2 X-ray diffraction 134
11.4 Results 134
11.4.1 MXT 134
11.4.2 XRD 136
11.5 Conclusions 138
CHAPTER 12: Comparison of Demineralisation results of HIMED and
PlASMA-BIOTAL Hydroxyapatite Discs
12.1 Aims and objectives 139
12.2 Materials and methods 139
12.2.1 SMR 139
12.2.2 HAp discs 139
12.2.3 Demineralisation solutions 140
12.3 Results 140
12.4 Conclusions 142
CHAPTER 13: Demineralisation of Compressed Hydroxyapatite Discs with
Acidic Buffer at a Range of pH Values Over Short Period of Time
13.1 Introduction 144
13.2 Aims and objectives 144
13.3 Materials and methods 145
13.3.1 SMR 145
13.3.2 HAp discs 145
TABLE OF CONTENTS
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13.3.3 Demineralisation solutions 145
13.4 Results 146
13.4.1 0.3% citric acid demineralisation solution 146
13.4.2 0.1% acetic acid demineralisation solution 149
13.5 Discussion 152
13.6 Conclusions 153
CHAPTER 14: The Effect of Demineralisation Solution on Compressed
Hydroxyapatite Discs Dissolution Studied Using Scanning Microradiography
14.1 Introduction 154
14.2 Aims and objectives 154
14.3 Materials and methods 155
14.3.1 SMR 155
14.3.2 HAp discs 155
14.3.3 Demineralisation solutions 155
14.3.4 Circulating pump 155
14.4 Results 158
14.5 Discussion 161
14.6 Conclusions 163
CHAPTER 15: The Effect of High Concentration of Strontium (Sr2+
) on
Hydroxyapatite Dissolution Kinetics Studied Using Scanning
Microradiography
15.1 Introduction 164
15.2 Aims and objectives 164
15.3 Materials and methods 165
15.3.1 HAp discs 165
15.3.2 Demineralisation solutions 165
15.3.3 SMR 165
15.4 Results 166
15.4.1 0.1% acetic acid pH4.0 with 6% strontium acetate 166
15.4.2 0.1% acetic acid pH4.0 with 8% strontium acetate 167
15.4.3 de-ionised water with 6% strontium acetate 168
TABLE OF CONTENTS
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15.4.4 de-ionised water with 8% strontium acetate 169
15.5 Discussion 169
15.6 Protocol summary 170
PART IV: EXPERIMENTAL WORK
CHAPTER 16: Effect of Zinc Ions (Zn2+
) on Hydroxyapatite Dissolution
Kinetics Studies Using Scanning Microradiography
16.1 Introduction 174
16.2 Aims and objectives 174
16.3 Materials and methods 175
16.3.1 HAp discs 175
16.3.2 Demineralisation solutions HAp discs 176
16.3.3 SMR 176
16.4 Results 177
16.4.1 0.1% acetic acid pH 4.0 177
16.4.2 0.3% citric acid pH 2.8 181
16.5 Discussion 185
16.6 Conclusions 192
CHAPTER 17: Effect of Strontium Ions (Sr2+
) at a Range of Concentrations
(0-30 ppm) on Hydroxyapatite Dissolution Kinetics Studied Using Scanning
Microradiography
17.1 Introduction 193
17.2 Aims and objectives 194
17.3 Materials and methods 194
17.3.1 HAp discs 195
17.3.2 Demineralisation solutions 195
17.3.3 SMR 195
17.4 Results 196
17.4.1 0.1% acetic acid pH 4.0 196
17.4.1 0.3% citric acid pH 2.8 200
17.5 Discussion 203
TABLE OF CONTENTS
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17.6 Conclusions 211
CHAPTER 18: : Effect of Copper Ions (Cu2+
) on Hydroxyapatite Dissolution Kinetics
18.1 Introduction 212
18.2 Aims and objectives 213
18.3 Materials and methods 213
18.3.1 HAp discs 214
18.3.2 Demineralisation solutions 214
18.3.3 SMR 214
18.4 Results 215
18.4.1 0.1% acetic acid pH 4.0 215
18.4.2 0.3% citric acid pH 2.8 219
18.5 Discussion 223
18.6 Conclusions 231
PART V: GENERAL DISCUSSION, CONCLUSIONS, CLINICAL
IMPLICATIONS AND RECOMMENDED FUTURE WORKS
CHAPTER 19: General Discussion, Conclusions, Clinical Implications, and
Recommended Future Works
19.1 General discussion 232
19.2 Conclusions 236
19.3 Clinical implication 237
19.3.1 Zinc 239
19.3.2 Strontium 240
19.3.3 Copper 241
19.4 Recommended future works 241
REFERENCES 244
APPENDIX I : ABSTRACTS FOR CONFERENCE PRESENTATIONS
AND PAPERS IN PREPARATION
256
LIST OF FIGURES
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List of figures
FIGURE 2.1 (a) Hexagonal unit structure of HAp with ions arranged
around the central hydroxyl column (c-axis). (b) Examples
of substitutes in biological apatite straining the lattice
parameters and changing the crystal behaviour (Robinson,
2000)
34
FIGURE 3.1 Schematic for enamel caries zones as classified by
Silverstone (1981)
41
FIGURE 3.2 Upper arch of child with gastro-oesophageal reflux showing
generalised erosion affecting maxillary teeth particularly on the
palatal surface (Welbury et al., 2005)
48
FIGURE 3.3 Schematics of citrate ion where two and three of the hydrogen
ions have been lost (a and b respectively) and calcium ion is
attracted (Lussi, 2006)
51
FIGURE 3.4 Dental erosion affecting both maxillary and mandibular teeth
particularly palatal and lingual surfaces (Lazarchik and Filler,
1997)
53
FIGURE 5.1 Schematic figure for the structure of Zn-doped HAp, where
yellow, blue, red, black, green and gray refer to calcium1
site,calcium2 site,oxygen, hydrogen, zinc and phosphate
groups respectively (Tang et al., 2009)
72
FIGURE 7.1 The effect of Cu2+
concentration on the phosphate released from
powdered human enamel (Brookes et al., 2003) after the
conversion of Cu2+
concentrations from mmol/L to ppm
86
FIGURE 8.1 X-ray as an electromagnetic wave, where the electric and
magnetic fields are perpendicular to each other and to the
direction of propagation (Seibert, 2004)
88
FIGURE 8.2 The electromagnetic spectrum in terms of wave length
(illustration from abrisa glass & coatings, 2005)
89
FIGURE 8.3 First X-ray photograph taken by Roentgen showing his
wife’s fingers (Assmus, 1995)
90
FIGURE 8.4 Schematic diagram showing basic components of an X-ray
tube (a) and X-ray tube used in SMR machine
(PANalytical®
) with silver (Ag) target (b)
91
FIGURE 8.5 A typical X-ray spectrum produced by a tube with tungsten
target showing continuous and characteristic radiation
93
FIGURE 8.6 Factors affecting the X-ray spectrum. (a) changing the tube
voltage changes the X-ray spectrum; (b) effect of tube
current on the X-ray spectrum; (c) effect of target material
96
LIST OF FIGURES
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on the spectrum; (d) adding a filter changes the shape of
the X-ray spectrum (Pobe, 1998)
FIGURE 8.7 X-ray attenuation mechanism: (a) Photoelectric effect; (b) Simple
scatter; (c) Compton scatter
99
FIGURE 8.8 Attenuation of a monochromatic X-ray beam of intensity I0
by a homogenous material thickness x
99
FIGURE 9.1 SMR machine with its main components X-ray source, X-
Y stage, and detector
104
FIGURE 9.2 Schematic representation of the SMR system main
components and their connections
104
FIGURE 9.3 Area scan of an SMR cell with the specimen centrally
located where X and Y axis represents specimen position
coordinates on the SMR stage. Two line scans drawn
across the specimen ( ) and scanning parameters are
shown on the side
107
FIGURE 9.4 Example of data analysis and construction of time profile of hap
mineral mass loss at the scanning positions during the
demineralisation process the error in each is of the order of 0.002
g/cm2
109
FIGURE 10.1 Schematic diagram of the cross section of the aperture assembly
D =10 µm ± 0.5 µm, L = 20 µm ± 1.0 µm
113
FIGURE 10.2 The main components of the SMR machine including the X-ray
source, X-ray detector, X-Y scanning stage, and the mounting
frame with SMR cells
114
FIGURE 10.3 Typical example of linear change in projected mineral mass
content over the experimental duration and the calculation of the
RDHAp
116
FIGURE 10.4 Change in the projected HAp hap mineral mass content over 24 h
at 100% sampling frequency
117
FIGURE 10.5 Change in the projected HAp mineral mass content over 24 h at
50% sampling frequency
118
FIGURE 10.6 Change in the projected HAp mineral mass content over 24 h at
25% sampling frequency time
118
FIGURE 10.7 Change in the projected HAp mineral mass content over 24 h at
10% sampling frequency
119
FIGURE 10.8 Change in the projected HAp mineral mass content over 24 h at
100% sampling frequency
121
FIGURE 10.9 Change in the projected HAp hap mineral mass content over 24 h 122
LIST OF FIGURES
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at 50% sampling frequency
FIGURE 10.10 Change in the projected HAp mineral mass content over 24 h at
33% sampling frequency
122
FIGURE 10.11 Change in the projected HAp mineral mass content over 24 h at
25% sampling frequency
123
FIGURE 10.12 Schematic diagram showing top and side views of the new design
for SMR cells with dimensions 124
FIGURE 10.13 New SMR cell design developed to accommodate fitting the
complete HAp disc required in this thesis
125
FIGURE 11.1 HIMED and Plasma-Biotal HAp discs placed flat and fixed
on a Perspex stand with aluminum wire to be mounted on
XMT rotation stage
133
FIGURE 11.2 Reconstructed images of coronal sections through two
compressed HAp discs showing larger pores in upper HAp
disc (HIMED) and evenly distributed and sized pores in
lower HAp disc (Plasma- Biotal)
135
FIGURE 11.3 Reconstructed images of axial sections through 4 HAp
discs top two and lower right discs (HIMED) showing
uneven distribution of larger sized pores while lower left
disc (Plasma-Biotal) shows even distribution of equally
sized pores
135
FIGURE 11.4 XRD pattern for HIMED HAp disc from 20–40 (2)
136
FIGURE 11.5 XRD pattern for Plasma-Biotal HAp disc from 20-40 (2) 137
FIGURE 11.6 Typical XRD pattern of fully crystalline HAp with principal
diffraction peaks (Prevéy, 2000)
137
FIGURE 12.1 The change in RDHAp for Plasma-Biotal and HIMED HAp
discs as a function of 0.1% acetic acid at a range of pH values
141
FIGURE 12.2 The change in RDHAp for Plasma-Biotal and HIMED HAp
discs as a function of 0.3% citric acid at a range of pH values
141
FIGURE 13.1 The change in HAp disc mineral mass content in response to 20 h
demineralisation by 0.3% citric acid pH 3.2 followed by 4 h of
de-ionised water
146
FIGURE 13.2 The change in HAp disc mineral mass content in response to 20 h
demineralisation by 0.3% citric acid pH 3.2 followed by 4 h of
de-ionised water
147
FIGURE 13.3 The change in projected HAp mineral mass content in response to
20 h demineralisation by 0.3% citric acid pH 3.6 followed by 4 h
of de-ionised water
147
LIST OF FIGURES
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FIGURE 13.4 The change in projected HAp mineral mass content in response to
20 h demineralisation by 0.3% citric acid pH 4.0 followed by 4 h
of de-ionised water
148
FIGURE 13.5 The change in projected HAp mineral mass content in
response to 20 h demineralisation by 0.1% acetic acid pH
2.8 followed by 4 h of de-ionised water
149
FIGURE 13.6 The change in projected HAp mineral mass content in
response to 20 h demineralisation by 0.1% acetic acid pH
3.2 followed by 4 h of de-ionised water
149
FIGURE 13.7 The change in projected HAp mineral mass content in
response to 20 h demineralisation by 0.1% acetic acid pH
3.6 followed by 4 h of de-ionised water
150
FIGURE 13.8 The change in projected HAp mineral mass content in
response to 20 h demineralisation by 0.1% acetic acid pH
4.0 followed by 4 h of de-ionised water
150
FIGURE 13.9 The change in RDHAp in response to changing the
demineralisation solution at a range of pH values
151
FIGURE 13.10 The change in RDHAp in response to changing the
demineralisation solution at a range of [H+]
151
FIGURE 14.1 Watson Marlow 205U electric pump with circulating
solution
156
FIGURE 14.2 The electric pump connected to the SMR cells via tubing while
demineralisation solution is circulating into and out of the SMR
cells
156
FIGURE 14.3 Typical example of the change in projected HAp mineral mass
content over a period of 24 h in response to 0.1% acetic acid pH
4.0 demineralisation solution at 0 ml/min circulation rate.
159
FIGURE 14.4 Typical example of the change in projected HAp mineral mass
content over a period of 24 h in response to 0.1% acetic acid pH
4.0 demineralisation solution at 0.97 ml/min circulation rate
160
FIGURE 14.5 The mean rate of demineralisation (g/cm2/h) plotted
against the change in demineralisation solution circulation
speed (RPM). A curve has been fitted for viewing purposes
only
161
FIGURE 15.1 Increased projected HAp mineral mass content over a
period of 40 h in response to exposure to 0.1% acetic acid
pH 4.0 demineralisation solution containing 6% strontium
acetate
166
FIGURE 15.1 Increased projected HAp mineral mass content over a
period of 40 h in response to exposure to 0.1% acetic acid
pH 4.0 demineralisation solution containing 8% strontium
acetate
167
LIST OF FIGURES
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FIGURE 15.3 Increased projected HAp mineral mass content over a
period of 40 h in response to exposure to de-ionised water
pH7 containing 6% strontium acetate
168
FIGURE 15.4 Increased projected HAp mineral mass content over a
period of 40 h in response to exposure to de-ionised water
pH7 containing 8% strontium acetate
169
FIGURE 16.1 Schematic diagram of an SMR cell with HAp disc in place
connected to the peristaltic pump (p) for circulating the
demineralisation solution over a period of 20 h followed by 30
minutes of de-ionised water
175
FIGURE 16.2 Typical example of the change in projected HAp mineral mass
content over a period of 20 h in response to 0.1% acetic acid pH
4.0 with 5 ppm Zn2+
demineralisation solution at increasing Zn2+
concentration sequence.
178
FIGURE 16.3 Typical example of the change in projected HAp mineral mass
content over a period of 20 h in response to 0.1% acetic acid pH
4.0 with 5 ppm Zn2+
demineralisation solution at decreasing Zn2+
concentration sequence
179
FIGURE 16.4 Typical example of the change in projected HAp mineral mass
content over a period of 20 h in response to 0.3% citric acid pH
2.8 with 5 ppm Zn2+
demineralisation solution at increasing Zn2+
concentration sequence
182
FIGURE 16.5 Typical example of the change in projected HAp mineral mass
content over a period of 20 h in response to 0.3% citric acid pH
2.8 with 5 ppm Zn2+
demineralisation solution at decreasing Zn2+
concentration sequence
183
FIGURE 16.6 The effect of Zn2+
at a range of 0–20 ppm on mean RDHAp in
increasing Zn2+
concentration sequence under caries-like
conditions
187
FIGURE 16.7 The effect of Zn2+
at a range of 20-0 ppm on mean RDHAp in
decreasing Zn2+
concentration sequence under caries-like
conditions
187
FIGURE 16.8 The effect of 0.1% acetic acid pH 4.0 with different Zn2+
concentration (ppm) on RDHAp (g/cm2/h) at both increasing
and decreasing concentration sequence
188
FIGURE 16.9 The effect of Zn2+
at a range of 0–20 ppm on mean RDHAp in
increasing Zn2+
concentration sequence under erosion-like
conditions.
190
FIGURE 16.10 The effect of Zn2+
at a range of 0–20 ppm on mean RDHAp in
increasing Zn2+
concentration sequence under erosion-like
conditions
190
FIGURE 16.11 The effect of 0.3% citric acid pH 2.8 with different Zn2+
191
LIST OF FIGURES
- 20 -
concentration (ppm) on RDHAp (g/cm2/h) at both increasing
and decreasing concentration sequence
FIGURE 17.1 Schematic diagram of an SMR cell with HAp disc in place,
connected to the peristaltic pump (p) for circulating the
demineralisation solution over a period of 20 hours
followed by 30 minutes of de-ionised water
194
FIGURE 17.2 Typical example of the change in projected HAp mineral mass
content over a period of ≈ 20 h in response to 0.1% acetic acid
pH 4.0 with 20 ppm Sr2+
demineralisation solution at increasing
Sr2+
concentration sequence
197
FIGURE 17.3 Typical example of the change in projected HAp mineral mass
content over a period of 20 h in response to 0.1% acetic acid pH
4.0 with 20 ppm Sr2+
demineralisation solution at decreasing Sr2+
concentration sequence
198
FIGURE 17.4 Typical example of the change in projected HAp mineral mass
content over a period of 20 h in response to 0.3% citric acid pH
2.8 with 20 ppm Sr2+
demineralisation solution at increasing Sr2+
concentration sequence
200
FIGURE 17.5 Typical example of te change in projected HAp mineral mass
content over a period of 20 h in response to 0.3% citric acid pH
2.8 with 20 ppm Sr2+
demineralisation solution at decreasing Sr2+
concentration sequence
201
FIGURE 17.6 The effect of Sr2+
at a range of 30-0 ppm on mean RDHAp at
increasing Sr2+
concentration sequence under caries-like
conditions
205
FIGURE 17.7 The effect of Sr2+
at a range of 30-0 ppm on mean RDHAp at
decreasing Sr2+
concentration sequence under caries-like
conditions
205
FIGURE 17.8 The effect of 0.1% acetic acid pH 4.0 with different Sr2+
concentrations (ppm) on RDHAp (g/cm2/h) at both increasing and
decreasing concentrations sequences
206
FIGURE 17.9
The effect of Sr2+
at a range of 0-30 ppm on mean RDHAp at
increasing Sr2+
concentration sequence under erosion-like
conditions
209
FIGURE 17.10
The effect of Sr2+
at a range of 0-30 ppm on mean RDHAp in
decreasing Sr2+
concentration sequence under erosion-like
conditions
209
FIGURE 17.11
The effect of 0.3% citric acid pH 2.8 with different Sr2+
concentrations (ppm) on RDHAp (g/cm2/h) at both increasing and
decreasing concentrations sequences
210
FIGURE 18.1 Schematic diagram of an SMR cell with HAp disc in place
connected to the peristaltic pump (p) for circulating the 213
LIST OF FIGURES
- 21 -
demineralisation solution over a period of 20 h followed by 30
minutes of de-ionised water
FIGURE 18.2 Typical example of the change in projected HAp mineral mass
content over a period of 20 h in response to 0.1% acetic acid pH
4.0 with 22.5 ppm Cu2+
demineralisation solution at increasing
Cu2+
concentration sequence
216
FIGURE 18.3 Typical example of the change in projected HAp mineral mass
content over a period of 20 h in response to 0.1% acetic acid pH
4.0 with 22.5 ppm Cu2+
demineralisation solution at decreasing
Cu2+
concentration sequence
217
FIGURE 18.4 Typical example of the change in projected HAp mineral mass
content over a period of 20 h in response to 0.3% citric acid pH
2.8 with 22.5 ppm Cu2+
demineralisation solution at increasing
Cu2+
concentration sequence
220
FIGURE 18.5 Typical example of the change in projected HAp mineral mass
content over a period of 20 h in response to 0.3% citric acid pH
2.8 with 22.5 ppm Cu2+
demineralisation solution at increasing
Cu2+
concentration sequence
221
FIGURE 18.6 The effect of Cu2+
at a range of 0–180 ppm on mean RDHAp at
increasing Cu2+
concentration sequence under caries-like
conditions
225
FIGURE 18.7 The effect of Cu2+
at a range of 180-0 ppm on mean RDHAp at
decreasing Cu2+
concentration sequence under caries-like
conditions
225
FIGURE 18.8 (a)The effect of Cu2+
concentration on phosphate released from
powdered enamel as published by Brookes et al.(2003) after the
conversion of mmol/L to ppm; (b) example of the effect of Cu2+
at a range of 0-180 ppm on mean RDHAp as observed in this study
226
FIGURE 18.9 The effect of 0.1% acetic acid pH 4.0 with different Cu2+
concentrations (ppm) on RDHAp (g/cm2/h) at both increasing and
decreasing concentrations sequences
227
FIGURE 18.10 The effect of Cu2+
at a range of 0–180 ppm on mean RDHAp at
increasing Cu2+
concentration sequence under erosion-like
conditions
229
FIGURE 18.11 The effect of Cu2+
at a range of 180–0 ppm on mean RDHAp at
increasing Zn2+
concentration sequence under erosion-like
conditions
229
FIGURE 18.12 The effect of 0.1% acetic acid pH 4.0 with different Cu2+
concentrations (ppm) on RDHAp (g/cm2/h) at both increasing and
decreasing concentrations sequences
230
LIST OF TABLES
- 22 -
List of tables
TABLE 3.1 Eccles and Jenkins erosion grading scale cited in (Lazarchik and
Filler, 1997)
53
TABLE 10.1 The RDHAp, R2 and SE calculated at different sampling
frequencies using Microsoft Office Excel 2003® and TableCurve
2D® programs
120
TABLE 10.2 The RDHAp, R2 and SE calculated at different sampling
frequencies representing different number of SMR cells scanned
simultaneously, using Microsoft Office Excel 2003® and
TableCurve 2D® programs
124
TABLE III.A Experiments performed for developing the thesis protocol
131
TABLE 12.1 RDHAp for both types of HAp discs in response to change
in demineralisation solution type and pH values
140
TABLE 14.1 Manufacturer tubes specifications and flow rate as factor
of change in pumping speed
156
TABLE 14.2 The measured flow rate in ml/min corresponding to each
circulating speed in RPM.
157
TABLE 14.3 The calculated RDHAP during the exposure to 0.1% acetic
acid pH 4.0 at various circulation speeds (in triplicate)
158
TABLE 14.4 Statistical analysis, for the data in Figure 14.3, using TableCurve
2D®
159
TABLE 14.5 Statistical analysis, for the data in Figure 14.4, using TableCurve
2D®
160
TABLE 15.1 A summary of the protocol to be used in the SMR studies
in this thesis
172
TABLE 16.1 Statistical analysis, for the data in Figure 16.2, using
TableCurve 2D®
178
TABLE 16.2 Statistical analysis, for the data in Figure 16.3, using
TableCurve 2D®
179
TABLE 16.3 RDHAp and calculated SE for each demineralising solution 180
TABLE 16.4
Statistical analysis, for the data in Figure 16.4, using TableCurve
2D®
182
TABLE 16.5 Statistical analysis, for the data in Figure 16.5, using TableCurve
2D
183
LIST OF TABLES
- 23 -
TABLE 16.6
RDHAp and calculated SE for each demineralisingsolution
184
TABLE 17.1 Statistical analysis, for the data in Figure 17.2, using TableCurve
2D®
197
TABLE 17.2 Statistical analysis, for the data in Figure 17.3, using TableCurve
2D®
198
TABLE 17.3 RDHAp and SE for each demineralisation solution at different
Sr2+
concentrations at both increasing and decreasing
concentration sequences
200
TABLE 17.4 Statistical analysis, for the data in Figure 17.4, using TableCurve
2D®
201
TABLE 17.5 Statistical analysis, for the data in Figure 17.5, using TableCurve
2D®
202
TABLE 17.6 The RDHAp and SE for each demineralisation solution at
different Sr2+
concentrations at both increasing and
decreasing concentration sequences
203
TABLE 18.1 Statistical analysis, for the data in Figure 18.2, using TableCurve
2D®
217
TABLE 18.2 Statistical analysis, for the data in Figure 18.3, using TableCurve
2D®
218
TABLE 18.3 RDHAp and SE for each demineralisation solution at
different Cu2+
concentrations at both increasing and
decreasing concentration sequences
219
TABLE 18.4 Statistical analysis, for the data in Figure 18.4, using TableCurve
2D®
221
TABLE 18.5 Statistical analysis, for the data in Figure 18.5, using TableCurve
2D®
222
TABLE 18.6 The RDHAp and SE for each demineralisation solution at
different Cu2+
concentrations at both increasing and
decreasing concentration sequences
223
LIST OF ABBREVIATIONS
- 24 -
List of abbreviations
a Intercept
Al Aluminium
°C Degree celsius
Ca2+
Calcium ion
CMR Conventional contact microradiography
Cu2+
Copper ion
DEJ Dentine enamel junction
DMFT Decayed, missing, filled permanent tooth
ESEM Environmental scanning electron microscopy
h Hour
H+ Hydrogen ion
HAp Hydroxyapatite
I Transmitted X-rays intensity
Io Incident of X-rays intensity
LAC Linear attenuation coefficient
m Mass per unit area
MAC Mass attenuation coefficient
MCA Multiple channel analyser
min Minute
RDHAp Hydroxyapatite demineralisation rate
s Seconds
SD
SE
Standard deviation
Standard error
SEM Scanning electron microscopy
SMR Scanning microradiography
Sr2+
Strontium ion
WHO World Health Organisation
XRD X-ray diffraction
XMT X-ray microtomography
Zn2+
Zinc ion
µ LAC in cm-1
µm Mass attenuation coefficient
- 25 -
PART I: INTRODUCTION AND LITERATURE
REVIEW
PART I: INTRODUCTION AND LITERATURE REVIEW
- 26 -
CHAPTER 1
Introduction
1.1 General introduction
Dental caries is a result of mineral dissolution of dental hard tissue, caused by
the acid metabolic end products of oral bacteria that are capable of fermenting
carbohydrates, particularly sugars. It is a multifactorial process and the presence of
other factors, such as the host and enough time for the fermentation and acid
production to take place, is required for caries to develop.
Dental caries is a worldwide health problem affecting both industrial and
developing countries. According to Peterson (2003) approximately five billion
people worldwide have experienced dental caries. It continues to be a major problem
in dentistry and therefore should receive attention in everyday practice, not only
considering treatment and restorative aspects but also preventive aspect.
Dental erosion is the loss of tooth hard tissue caused by acids without
bacterial involvement. It is generally agreed that the reported prevalence of dental
erosion is increasing. This may be due to greater awareness of the condition among
dentists, and the increase in ageing populations worldwide, and the adult population
retaining more natural teeth as they age due to developments in dentistry and dental
care. In addition, younger individuals appear to exhibit increased dental erosion,
PART I: INTRODUCTION AND LITERATURE REVIEW
- 27 -
which may be due to more acidic diets and dietary eating disorders such as bulimia
and anorexia.
Although dental erosion is increasingly recognised as an important aetiology
in the loss of tooth structure, not only in adults but in adolescents and children as
well, little is established concerning diagnostic criteria, treatment and preventive
strategies. There is still a lot to be done in this field.
Mature dental enamel is acellular highly mineralised dental tissue that
consists mostly of impure forms of hydroxyapatite (HAp). Carbonate, sodium and
magnesium are the most abundant impurities; however a large number of impurities
may exist. These may alter the physical and chemical properties of HAp and
accordingly affect its demineralisation process. This thesis will address the effect of
three divalent cations, zinc (Zn2+
), strontium (Sr2+
) and copper (Cu2+
), on the HAp
demineralisation process under caries and erosion-like conditions in an attempt to
understand their effect on the kinetics of HAp demineralisation process and their
potential usefulness as a part of a preventive oral regimen against dental caries and
erosion.
In this thesis the technique used for studying the effect of divalent cations on
HAp demineralisation, is scanning microradiography (SMR). It is a method of
mineral quantification by means of X-ray absorption in which the radiographic
emulsion is replaced by a solid state detector. As part of the experimental work done
for this thesis, the standard SMR technique has been modified to allow reliable
quantitative data to be obtained over a short period of time (24 h or less), and the
newly developed technique has been used in all the studies in this thesis.
PART I: INTRODUCTION AND LITERATURE REVIEW
- 28 -
1.2 General aim
The general aim of this study was to investigate the effect of the divalent
cations zinc, strontium and copper on the physical chemistry influencing HAp
dissolution kinetics, using scanning microradiography under simulated cariogenic
and erosive conditions relevant to the oral environment.
1.3 Thesis layout
This thesis has been divided into four parts:
Part I, comprises the introduction to the thesis and the literature review. It is
divided into nine chapters. The first three chapters deal with the literature review of
dental enamel, dental caries and dental erosion with a brief overview of some of the
available dissolution models for calcium phosphates. Chapters 5, 6 and 7 contain a
detailed literature review of Zn2+
, Sr2+
, and Cu2+
respectively, as the divalent cations
of interest in this thesis. Chapter 8 and Chapter 9 are concerned with the review of
X-ray microradiography including X-ray generation, types of X-ray tubes, X-ray
interactions with matter, X-ray attenuation and X-ray detection. Finally, the last
chapter in Part I is a review of the literature on scanning microradiography as a
technique of interest to this thesis.
Part II contains the methodology. It describes in detail the modifications
made to the SMR technique, as part of the work in this thesis, so that it can be used
to produce a reliable quantitative data over a short period of time (24 h or less).
Part III describes the protocol development. It consists of five chapters
investigating the several changeable SMR parameters aimed at developing a protocol
to be used for the rest of the experiments in this thesis.
PART I: INTRODUCTION AND LITERATURE REVIEW
- 29 -
Part IV consists of three chapters investigating the three divalent cations.
Each chapter includes its own introduction, aims and objectives, materials and
methods, results and discussion.
Finally, the work presented in this thesis is collectively summarised and
addressed in Part V through an overall discussion, conclusions, discussion of the
clinical implications and recommendations for future work.
PART I: INTRODUCTION AND LITERATURE REVIEW
- 30 -
CHAPTER 2
Human Dental Enamel
2.1 Dental enamel chemical composition
Dental enamel is a highly mineralised acellular dental tissue that is often
referred to as an inorganic-organic two-phase system. It consists of ≈ 98 wt.% or 96
volume % calcium HAp, with multiple impurities, and ≈ 2 wt.% organic matrix and
water (Elliott, 1994).
The organic matrix consists mainly of proteins. However, lipids,
carbohydrates, and other organic molecules are also present (Wilson et al., 1999).
The protein concentration in dental enamel varies in a systematic manner. A high
concentration of proteins has been reported to be located at the inner enamel of
fissures and at the cervical margins (Robinson et al., 1983).
The inorganic components are mainly in the form of impure HAp.
Hydroxyapatite is a naturally occurring mineral with the chemical formula
Ca5(PO4)3(OH), but now usually written as the stoichiometrically correct atomic
composition containing 10 calcium atoms: Ca10(PO4)6(OH)2. Inclusion of carbonate,
sodium, fluoride and other ions result in the impure form of the HAp that is present
in human dental enamel (Elliott, 1997). In enamel crystal, phosphate ions can be
replaced by carbonate ions, calcium ions can be replaced by sodium, and hydroxyl
ions can be replaced by fluoride ions. Although there is no limit to the possible
PART I: INTRODUCTION AND LITERATURE REVIEW
- 31 -
extent of this substitution, 100% replacement is very rare. For example a 100%
substitution of hydroxyl ions by fluoride ions lead to the formation of fluorapatite
which is rarely found in biological tissue (except in shark enameloid) (Elliott, 1994).
Substitution and distribution of some common impurities will be discussed in detail
in Section 2.4.
2.2 Dental enamel structure
The basic structural units of human enamel are ≈5 µm wide enamel rods or
sometimes referred to as enamel prisms (Boyde, 1997). Enamel rods extend from the
enamel-dentine junction to the tooth surface and are separated by the interrod region.
Each enamel rod is formed by tightly compacted highly organised enamel mineral
crystals (crystallites). The mature enamel crystallites are narrow crystals with
flattened hexagonal cross section (≈30 to 50 nm in width and elongated along the c-
axis) (Boyde et al., 1988). In cross section, the enamel rods may be compared to a
keyhole with the top, or head, oriented toward the crown of the tooth and the tail,
oriented toward the root of the tooth. The angle at which the rods approach the
enamel surface varies from 90° in the cervical region to approximately 10° in the
cuspal region. Many authors like Ripa et al. (1966), Whittaker (1982), Shellis
(1984), Kodaka et al. (1989) and Kodaka et al. (1991) have reported that unlike the
enamel bulk, surface enamel is prismless. The crystallites at the outer enamel are
aligned parallel to each other and perpendicular to the enamel surface resulting in a
more mineralised and densely packed layer with lack of inter-rod space.
PART I: INTRODUCTION AND LITERATURE REVIEW
- 32 -
2.3 Physical properties of dental enamel
Through crystallographic work Brudevold et al. (1960) concluded that the
composition of enamel crystal is of pure HAp and therefore the mineral density of
enamel would be equal to that of HAp (≈ 3.15 g/cm3). However, later studies (Elliott,
1997) showed that enamel consists mainly of the impure form of HAp with multiple
impurities, particularly carbonate ions that partially replace the phosphate ions. This
significantly reduces enamel density (between 2.99 and 3.02 g/cm3). Even though
enamel density is less than was previously thought, still dental enamel is considered
very dense and rigid material. The high rigidity and density makes it very brittle
unless supported by the underlying dentine.
Another characteristic feature of enamel that affects its physical properties is
enamel pores, which result from the imperfections in the packing of enamel
crystallites. They are usually filled or partially filled with inter-prismatic substance.
Authors have classified enamel pores into three main categories (Boyde and Oksche,
1989, Shellis and Dibdin, 2000). The first type is the small hexagonal tubule like
pores (1-10 nm in diameter). They are located within the body of the enamel prism
due to the random crystal orientation around the c-axis. They are the more abundant
type of pores and count for 1-5 vol% of enamel. The second type is the prisms
junctions pores. They are the largest in size but fewer in number and represent a
minor fraction of the total enamel porosity. The third type is the intra-prismatic but
their porosity is difficult to measure and little is known about them.
As a result of enamel structural architecture, particularly porosity, dental
enamel is considered permeable to water, ions and small size organic molecules. The
diffusion of water, ions and small organic molecules is controlled by many factors.
Principally they are controlled by pore number, pore size and the inter-connectivity
PART I: INTRODUCTION AND LITERATURE REVIEW
- 33 -
between the pores. The partial acceptance or rejection of ion transport through the
enamel pores depending on the charge of the diffusing ions is another controlling
factor. To a lesser extent; the organic matrix also plays a role in affecting the
permeability and transport process through enamel. For example, protein in the
enamel matrix limits ionic diffusion. Also the mobility of water through enamel
pores is significantly affected by the hydration of proteins (Shellis and Dibdin,
2000).
2.4 Trace elements in dental enamel
Dental enamel is composed mostly of biological apatites. They are impure
form of HAp and differ from HAp in their composition, crystal size, morphology and
stoichiometry. For example the Ca:P molar ratio in dental enamel is 1.62 - 1.64
while the Ca:P molar ratio in pure HAp is 1.67. This leads to the general idea that
biological apatites are calcium deficient or non-stoichiometric. Pure HAp consists of
calcium, phosphate and hydroxyl ions (Figure 2.1(a)) while biological apatites
contain small amounts of various trace elements such as CO32-
, Mg2+
, Na+, F
-, Zn
2+,
Cu2+
, Sr2+
and others in addition to the main components, Ca2+
, PO43-
, and OH-
(Figure 2.1(b)).
PART I: INTRODUCTION AND LITERATURE REVIEW
- 34 -
FIGURE 2.1 (a) Hexagonal unit structure of HAp with ions arranged around the
central hydroxyl column (c-axis). (b) Examples of substitutes in biological apatite
straining the lattice parameters and changing the crystal behaviour (schematic
drawing idea after Robinson (2000))
Once the anions or cations become incorporated into the apatite structural
lattice they alter the physico-chemical properties of the apatite. Such changes involve
changes in crystal lattice parameters (reflecting the size and amount of substituents),
change in crystallinity (crystal size and strain), change in crystal morphology and
change in dissolution properties. The following section discusses some common
substituents in dental enamel.
2.4.1 Carbonate
There has been controversy about the carbonate (CO32-
) substitution site in
the apatite lattice. However, there is now general agreement that carbonates can
either substitute for the phosphate ions which is called the B-type substitution
(LeGeros and Tung, 1983) or substitute for the hydroxyl group which is called the A-
type substitution (Elliott et al., 1985). Carbonates poorly fit into the HAp lattice
causing lattice strain and accordingly more soluble crystals. This is typically
illustrated in the A-type substitution, when the hydroxyl group is substituted by less
well-fitting carbonate which weakens the core of the crystal lattice along the c-axis.
(a) (b)
PART I: INTRODUCTION AND LITERATURE REVIEW
- 35 -
The weak central core has been suggested to be responsible for the greater solubility
of the crystals at the centre (Marshall and Lawless, 1981).
B-type substitution is usually associated with sodium ion replacement for
calcium. Therefore, the sodium concentration of the lattice is considered an indirect
indicator of carbonate concentration.
Like many other elements, carbonate distribution and concentration vary
throughout enamel thickness, with increasing concentration from the surface (1 wt%)
towards the inner enamel (4 wt%)(Robinson, 2000).
2.4.2 Fluoride
Fluoride can substitute in the apatite crystal either as F
- or CO3F3
- by filling
hydroxyl vacancies or by substituting the hydroxyl ion (Elliott, 1994). When fluoride
ion (ionic radius ≈ 1.36Ǻ) substitutes the hydroxyl ion (ionic radius, 1.40Ǻ) on the c-
axis it causes a reduction in the crystal volume and the lattice becomes more dense
which reduces the crystal dissolution constant and enhances its chemical stability
(Aoba, 1997). This substitution involves reduction at both the a and the c-axis (Kay
et al., 1964) and reduces the lattice energy bringing more stability to the lattice
(Robinson et al., 1995b).
Unlike carbonate, fluoride shows a higher distribution concentration at the
outer enamel surface than the inner enamel(Robinson, 2000).
2.4.3 Magnesium
Magnesium is considered a principal minor constituent of biological apatite.
There is uncertainty about the incorporation of magnesium in the HAp lattice
(Verbeeck, 1986). It has been reported that magnesium can substitute for calcium
ions. However this is a very minimal substitution as only a small amount of
PART I: INTRODUCTION AND LITERATURE REVIEW
- 36 -
magnesium can be accommodated in the HAp crystal lattice (0.3 wt%) (Featherstone
et al., 1983b). Another possibility is that magnesium adheres to the crystal surface
layer, either as an adsorbed element on the surface or attached loosely in the
hydration layer, rather than being incorporated in the structure (Robinson, 2000).
Like carbonates, magnesium shows higher concentrations in the inner enamel layer
than in the outer surface (Robinson, 2000).
In conclusion, the topic of structure, chemistry and properties of enamel
apatite has received a lot of attention from researchers and lots of fundamental work
has been published in this area including published textbooks and review papers such
as LeGeros (1991), Ten Cate and Featherstone (1991), Johnsson and Nancollas
(1992), Elliott (1994), Shellis and Duckworth (1994) and Aoba (1997). It is
particularly important to remember that dental enamel mineral contains not only
HAp, but an apatite like structure with a wide variety of substitutes that might alter
its physico-chemical properties. Zinc, strontium and copper as divalent metal cations
are of special interest to this thesis. Their effect will be discussed in details in
Chapter 5, 6 and 7 respectively.
2.5 Hydroxyapatite as a model system for dental enamel
Hydroxyapatite is commonly used as laboratory and, to a lesser extent,
mathematical model for dental enamel mineral. However, there is still some
controversy as to whether HAp can be used as a good representative of dental
enamel mineral.
In this section a brief over view of the similarities and differences between
HAp and dental enamel is discussed.
PART I: INTRODUCTION AND LITERATURE REVIEW
- 37 -
1. Crystal lattice parameter: the mineral of enamel has a different crystal lattice
parameter (spacing) from HAp. According to crystallographic studies for
HAp, a = 9.418 Ǻ and c = 6.881 Ǻ, while for dental enamel, a = 9.455 Ǻ and
c = 6.881 Ǻ (Wilson et al., 1999).
2. Chemical composition: HAp has a constant composition that can be
summarised in the chemical formula Ca10(PO4)6(OH)2 while dental enamel
has variable chemical composition with various impurities such as CO32-
2 to
4 wt% replacing PO43-
and Na+ 0.25 to 0.9 wt% (Section 2.3).
3. Density: due to the difference in chemical composition, enamel has a lower
mineral density (2.99-3.02 g/cm3) compared to the mineral density of HAp
(3.15 g/cm3).
4. Porosity: HAp typically has higher pores percentage, but pores are more
evenly sized and distributed, while dental enamel has overall lower porosity.
Pores size and distribution not only varies in dental enamel of different teeth,
they even vary between different areas in the same tooth.
Even though HAp and dental enamel minerals differ in some aspects, HAp is
still generally accepted as representative of dental enamel, and presents several
significant advantages. From the practical point of view HAp is considered
convenient to use as it is easier to obtain and requires no ethical approval.
Further, HAp has a well-defined chemical composition and density when
compared to enamel minerals. It also has the advantage of composition
adaptability as it can be chemically adapted by the addition of impurities such as
fluoride or sodium at precise levels of concentration, if needed, to mimic enamel
minerals. Synthetic sintered HAp allows the use of larger size samples and gives
reliable measurement repeatability due to its uniformity in chemical composition,
PART I: INTRODUCTION AND LITERATURE REVIEW
- 38 -
while for enamel minerals the repeatability of measurements is unreliable due to
structural variations. So in conclusion, HAp aggregates are not expected to react
identically to dental enamel as they are much more structurally and chemically
homogeneous than enamel, but are believed to exhibit very similar dissolution
kinetics and they can be used as a model for enamel in attempts to understand in
vivo caries or erosion formation (Shellis et al., 2010).
PART I: INTRODUCTION AND LITERATURE REVIEW
- 39 -
CHAPTER 3
Dental Enamel Caries and Erosion
3.1 Dental enamel caries
3.1.1 Introduction to dental enamel caries
Dental caries is the most common chronic disease affecting children (Filstrup
et al., 2003). It is five times more common than asthma (Donahue et al., 2005). Its
distribution varies between countries, regions within the same country as well as
social class and ethnic groups (Petersen, 2005, Christensen et al., 2010). According
to the National Survey of Children’s Health in the United Kingdom, almost 40% of
the 5 years old children in England and Wales in 2003 had dental caries (Pitts et al.,
2007).
Although the prevalence and extent of dental caries have fallen greatly in the
UK between the late 1970s and the current day, as well as in many other countries
such as Nordic countries and Switzerland, yet this decline seems to have slowed
down, and dental caries continues to be considered a significant problem.
According to the WHO 2003 report on oral health, caries remains a problem
despite great improvements in the dental public health (Petersen and Yamamoto,
2005). The report showed that caries has declined in many developed countries from
a decayed, missing and filled permanent teeth (DMFT) level of 4.5 to 2.5 for
children aged 12 years between the years 1980 and 1998, however, over the same
PART I: INTRODUCTION AND LITERATURE REVIEW
- 40 -
period of time the DMFT of the same age group increased from 1.5 to 2.5 in
developing countries. This is alarming considering that most of our world today is
made up of developing countries (Sgan-Cohen and Mann, 2007).
Therefore, dental caries is still considered a problem worth manageing
particularly through well-planned comprehensive dental health promotion and
preventive strategies.
3.1.2 Aetiology of dental caries
For as long as the science of dentistry has existed, there have been theories
about the causes of dental caries. However, today all experts in cariology generally
agree that dental caries is a complicated multifactorial process that leads to
destruction of dental hard tissue and that it is a localised destruction of dental hard
tissue caused by acids produced by dental plaque bacteria (Fejerskov et al., 2008). It
can take place on any tooth surface in the oral cavity when dental plaque is left to
accumulate for enough time to allow its bacteria to ferment the dietary carbohydrate
(Kidd and Fejerskov, 2004). Bacterial carbohydrate fermentation results in acid
production, such as, lactic acid, acetic acid, etc, which reduces the dental plaque pH
below 5.0 within 1-3 minutes (Kidd, 2005). Exposure of tooth surface to repeated
attacks of low pH may result in demineralisation. However, when the acid produced
in dental plaque is neutralised by saliva, the pH increases again and minerals may be
regained and remineralisation occur.
The cumulative result of the de- and remineralisation attacks determine
whether the tooth will undergo demineralisation or remineralisation (Aoba, 2004).
The process of demineralisation or remineralisation takes place frequently during the
day leading to cavitation, repair or a maintenance state.
PART I: INTRODUCTION AND LITERATURE REVIEW
- 41 -
However dental caries is not only an infectious disease induced by diet. It is a
complicated multifactorial process with multiple factors affecting the initiation and
progression of the disease. There are factors that directly contribute to caries
development. These include a host, dietary substrate, bacteria and sufficient time
frame. Oral environmental factors include, saliva buffering capacity, salivary
composition and flow rate, sugar consumption, frequency and sugar clearance rate.
Also important are plaque pH, types of microbial species, and the use of fissure
sealant, antimicrobial agents and fluoride. Finally, relevant personal factors include
the level of education, behaviour and attitude towards oral care, sociodemographic
status and many others (Harris et al., 2004).
Dental caries is recognised as a preventable disease. Furthermore, it is known
that cavitation is quite a late stage in the disease development and that before
cavitation; the progress of the disease may be arrested or reversed if a favourable
oral environment is achieved.
3.1.3 Histology and chemical changes in enamel caries
Silverstone (1981) has studied the histological changes of enamel in carious
lesions and divided them into four zones, starting from the outer enamel surface
layer to the enamel dentine junction (EDJ). These four zones are: surface, body of
the lesion (25-50%), dark (5-10%) and translucent (Figure 3.1).
FIGURE 3.1 Schematic for enamel caries zones as classified by Silverstone (1981)
PART I: INTRODUCTION AND LITERATURE REVIEW
- 42 -
1. Surface Zone
The surface zone is the outermost zone, usually about 40 µm thick. During the
process of dental caries, acids produced by bacteria diffuse into enamel and decrease
its pH which starts the demineralisation process. As a result of the decrease in pH
and the protonation of some phosphates (PO43-
) to hydrogen phosphates (HPO42-
),
apatite crystals become unstable. This step is described as the formation of an active
demineralisation site. As a result of the redistribution of charges and instability in the
apatite crystal bonds, calcium is released. The release of calcium and protonation of
phosphates, due to the drop in pH at the tooth outer surface, will form an
undersaturated layer, a principal requirement for mineral dissolution.
As the demineralisation process continues, more acids will continue to diffuse
inwards and more ions will be released and diffuse outwards. This outward and
inward exchange is a key model in describing enamel caries-subsurface
demineralisation. According to this theory, demineralisation starts at the subsurface
layer while the outer surface layer remains intact (Silverstone, 1981).The subsurface
demineralisation characteristic of dental enamel are cited in the literature to be due to
irregularities in structure, the organic matrix in dental enamel, or the presence of a
dental plaque layer (Isaac et al., 1958, Zahradnik and Moreno, 1977). However,
some in vitro studies on HAp aggregates have demonstrated subsurface
demineralisation. This indicates that subsurface demineralisation is a characteristic
of HAp rather than dental enamel (Mortimer and Tranter, 1971, Zahradnik et al.,
1976, Anderson and Elliott, 1985). Some models have been suggested in the
literature to explain the mechanism that relates inward and outward flux of ions
across the surface zone, such as the coupled diffusion model. The surface zone can
PART I: INTRODUCTION AND LITERATURE REVIEW
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be considered as the relatively intact layer of enamel with mineral mass loss of less
than 1%.
2. Translucent Zone
The enamel in this zone has more porosity and appears translucent when
embedded with Canada balsam and looked at under a light microscope (Silverstone,
1981). This zone shows a 10-fold increase in pore volume when compared to intact
enamel and accounts for approximately 1% of mineral loss, mostly mineral that is
rich in carbonate and magnesium (Robinson, 2000).
3. Dark Zone
If a tooth section is put into quinoline and viewed with polarised light the body
of the lesion will be outlined by a dark area (dark zone) (Kidd, 2005). The dark zone
looks dark because quinoline, being a large molecule, cannot get into the little holes,
which therefore remain filled with air giving a dark appearance while the body of
lesion which looks dark in water now looks translucent with quinoline (Ten Cate,
1998).
The dark zone is similar to the translucent zone as they both show porosity and
mineral loss, yet the dark zone shows mineral loss of about 5-10% and in addition to
the large pores seen in the translucent zone small pores are seen in the dark zone
(Robinson, 2000). The small pores in the dark zone show partial reversal of carious
lesions when exposed to saliva or synthetic calcifying solution in experiments. Some
studies (Crabb, 1966b, Crabb, 1966a, Silverstone, 1966, Clarkson et al., 1984,
Robinson et al., 1990) have shown that when artificial caries-like lesions are exposed
to saliva or synthetic calcifying solution, there is reduction in the pore volume
throughout the whole lesion. This suggests that the dark zone represents a zone
PART I: INTRODUCTION AND LITERATURE REVIEW
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where both demineralisation and remineralisation take place. This reflects the
dynamic nature of the caries process which involves episodes of demineralisation
and remineralisation simultaneously (Robinson, 2000).
Therefore, it has been suggested that the dark zone represent a dynamic stage
between demineralisation and remineralisation according to the surrounding
environment (Silverstone, 1981, Robinson et al., 1990, Robinson, 2000).
4. Body of Lesion
The body of the lesion is the main part of the lesion and considered as the final
stage of enamel demineralisation. The body of lesion is formed when the pore
volume is so great that there is a catastrophic collapse of the enamel structure,
followed by the collapse of the outer enamel surface layer (Robinson et al., 1983,
Shellis et al., 1993).
3.1.4 Methods of dental caries detection
Dental caries diagnosis is mostly carried out using visual examination of the
tooth surface with or without the use of a dental probe. This method of examination
is well established, however studies have shown that almost half of occlusal carious
lesions can be missed using this method of examination.
The use of the dental probe (explorer) in caries detection is controversial. In
the USA it is considered that a sharp explorer tip should be used to detect any
softness in the surface, while in Europe this practice is believed to add little benefit
to caries detection. On the contrary, it might cause iatrogenic damage to the enamel
surface and facilitate caries progression or initiation.
Proximal caries detection in posterior teeth can be challenging, especially in
cases of heavy contact. The use of dental wedges, orthodontic separators or trans-
PART I: INTRODUCTION AND LITERATURE REVIEW
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illumination might be of help. The use of dental radiographs is the method of choice
by most dentists. However, radiographs are not helpful in detecting caries at early
stages of development. In dental arches with crowding or rotated teeth the use of bite
wings become of very little value, so accordingly the use of radiographs become
more helpful in detecting advanced dentinal lesions.
Nowadays, a new caries detection and scoring system has been introduced, the
International Caries Detection and Assessment System (ICDAS) (Ismail et al.,
2007). It is a clinical scoring system that can be used for dental education, clinical
practice, research, and epidemiology (Pitts, 2004). It is designed to be based on a
better quality of collective information to achieve appropriate diagnosis, prognosis,
and clinical management at both the individual and public health levels. ICDAS has
the advantage of enabling personalisation of caries management for each case
independently, which helps in providing better and longer term results (Ismail et al.,
2008).
3.1.5 Prevalence of dental caries
In early 1900 the first statistics on dental decay were published (Yates, 1949,
Marthaler, 2004). That was approximately the time when the first university dental
faculties were training dental students. The number of these early statistics was very
low and they are difficult to interpret. Around the 1950s, indices and methods of
conducting surveys of dental diseases were developed, and in the 1960s many
epidemiological studies started.
Until the 1960s the published surveys suggested that the prevalence of dental
caries in children of Western European countries was high with an average of more
PART I: INTRODUCTION AND LITERATURE REVIEW
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than 5 DMFT for 12 year old children and 10 DMFT for 15 year old children
(Marthaler, 2004).
Between the 1970s and the 1980s there was a remarkable decline in the
prevalence of dental decay in children in many industrialised countries. This
reduction is mainly due to the development and the wide spread of use of fluoridated
tooth pastes (Downer et al., 1985, Downer, 1993).
During the decades since then, consensus from around the world shows that
dental caries has declined significantly. In 1985, FDI data demonstrated caries
declined particularly in nine countries: Denmark, Finland, Norway, Sweden,
Australia, the Netherlands, New Zealand, the United Kingdom and the USA
(Marthaler, 2004). In 12 year old children in the Netherlands the decrease in dental
caries showed the average DMFT decreased steadily from eight in 1965 to one in
1993. Similarly, most of the European data showed that caries prevalence in children
continued to decline until the 1990s (Downer et al., 1985). Although the last
National Children’s Dental Health Survey in the UK in 2003 showed that overall
dental caries in children continued to decline over the last decade yet there was an
observation of an increase in caries prevalence among particular groups such as the
lower social classes and migrants (Harker and Morris, 2005). This is shown in a
Swedish study, with Turkish immigrant children having more caries than Swedish
children both in the primary and permanent teeth (Mejàre and Mjönes, 1989).
However, children born in Turkey had more caries in the primary dentition than
those born in Sweden. Turkish immigrant children therefore constitute a high risk
group for caries and need supervision early after immigration. Also, increasing
immigration has been identified as a new factor, leading to increases in the overall
PART I: INTRODUCTION AND LITERATURE REVIEW
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dental caries prevalence rate in Switzerland, given that migrants form 20% of Swiss
residents.
Most recent studies have reported that dental caries is increasing particularly
in developing countries. This is alarming given that most of today’s world
population is made of developing countries.
The National Oral Health Survey in the Philippines reported an alarming
97.1% of 6 year old with dental caries and 84.7% with symptoms of dental infection.
The overall prevalence of dental caries among 6-12 year old school children was
92.3% (Carino et al., 2003). In Mexico, the prevalence of dental caries increased by
more than 20% among children in just over one year from 14.2% to 34.7% in fewer
than 18 months. An epidemiological survey in Sao Paulo, Brazil showed that the
prevalence of dental caries in permanent teeth among 12 year old children was
53.6% (Gomes et al., 2004). In Palestine, the DMFT score was 6.5 in an oral health
survey (Bagramian et al., 2009). In Saudi Arabia there is lack of national oral health
survey. However local and regional surveys reported a high DMFT score in 12 years
old children. In Riyadh area for example the mean DMFT was 5.06 (AlDosari et al.,
2004). Another study conducted in the western region (Jeddah) reported a mean
DMFT of 5.71(Alamoudi et al., 1996).
In summary dental caries remains a major health concern worldwide and an
action is needed to control the spread of this problem.
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3.2 Dental erosion
3.2.1 Introduction to dental erosion
Dentists have been aware of the phenomenon of dental hard tissue loss that is
not attributed to dental caries but for years only little has been done about it.
Recently, such dental hard tissue loss has been increasingly seen in the younger
population (Welbury et al., 2005). The phenomenon of tooth wear can be classified
as attrition, abrasion or erosion. Attrition is loss of the tooth hard surface due to tooth
to tooth contact (bruxism). Abrasion is physical wear due to tooth surface contact
against hard surfaces such as a faulty brushing technique with a hard toothbrush or
the habit of nail biting or biting against a pen or pencil while thinking. Erosion can
be defined as the loss of dental hard tissue due to acids without the involvement of
bacteria (Figure 3.2).
FIGURE 3.2 Upper arch of child with gastro-oesophageal reflux showing generalised
erosion affecting maxillary teeth particularly on the palatal surface (Welbury et al.,
2005)
PART I: INTRODUCTION AND LITERATURE REVIEW
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3.2.2 Aetiology of dental erosion
Erosion can be due to intrinsic factors or extrinsic factors. For example the
pH of stomach acid can reach below 1.0 and therefore any regurgitation or vomiting
is harmful to the teeth and causes more severe destruction than that caused by other
dietary acids (Bartlett and Coward, 2001). Gastro-intestinal tract disorders or eating
disorders (e.g. bulimia and anorexia nervosa) are the most common causes of dental
erosion by gastric acid (Meurman et al., 1994, Schroeder et al., 1995). However,
extrinsic factors are considered the most common cause of dental erosion. Extrinsic
factors are most commonly in the form of acidic foods or drinks such as fruit, fruit
juices, carbonated drinks, and sports drinks. Many of these acids are usually
unnoticed by their consumers and their effect is underestimated (Gandara and
Truelove, 1999). Pure baby fruit juices, for example, have been shown to have a pH
value below 5.5. Many of these drinks are given to infants in a feeding bottle and the
combination of the prolonged exposure of the tooth to the juice and its highly acidic
nature may result in excessive tooth surface loss (Zerob, 2004). Soft drinks represent
a major factor of dental erosion through their ability to cause enamel and dentin
dissolution, and they are in particular available to all age groups (Nyvad, 1999). In
1995, one study showed that 56-85% of USA school children consumed at least one
soft drink per day, from this group 20% consumed four or more servings daily
(Grenby, 1996). Although the nature of the acidic food or drink has a strong effect
on the degree of dental erosion, it is not the only controlling factor (Amaechi and
Higham, 2005).
It was found that the volume, frequency and time of consumption affect the
degree of dental erosion as erosive tooth surface loss tends to be higher in cases of
high volume of consumption and when the intake is at bed time (Moazzez et al.,
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2000). Behavioural factors can influence the impact of these dietary acids on the
dentition. For example, excessive consumption of acidic food or beverages, or
unusual eating and drinking habits such as sipping an acidic drink over a long period
of time, will increase the acid challenge to the teeth (Johansson et al., 2004). Other
acidic foods and drinks such as wine, and vinegar are potentially erosive (Chaudhry
et al., 1997, Piekarz et al., 2008). The most commonly found acids in soft drinks are;
citric, phosphoric, malic and tartaric acids (Grenby, 1996). A study of sour sweets,
which are popular among children came to an important conclusion: that all the sour
sweets tested were found to be erosive, and some of them were even more erosive
than orange juice (Chu et al., 2010). This is important to know, especially for
paediatricians and paediatric dentists who are concerned about children’s dietary
habits and diet analysis (Chadwick, 2008, Brand et al., 2009, Wagoner et al., 2009).
Oral hygiene products such as toothpastes, and some low pH medications, like
vitamin C tablets, have been reported to show erosive potential (Lussi, 2006).
Environmental acids are also potential risk factors. Acidic fumes such as sulfuric and
hydrochloric acid fumes in some working places have been reported to show erosive
potential (Petersen and Gormsen, 1991).
Dental erosion can be clinically observed at early stages of development as a
loss of surface contour with a shiny, glass like appearance (Asher and Read, 1987).
In the past it was thought that erosion involved the total loss and destruction of the
whole enamel thickness while some studies have demonstrated signs of subsurface
demineralisation (Meurman and Gate, 1996). Therefore the chemical processes of
dental enamel erosion and dental enamel caries are quite similar, apart from the
source of acids and the lack of dark zone. The absence of dark zone might be due to
the very low pH in the case of erosion. Lussi and Featherstone have studied the
PART I: INTRODUCTION AND LITERATURE REVIEW
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chemistry of dental erosion (Lussi, 2006). A key factor in dental erosion is that it
takes place in a highly acidic environment and the mineral loss can be a result of
simple interaction with hydrogen ions such as in the case of acetic acid.
CH3COOH↔ CH3COO-
+ H+
(3.1)
However, it is more likely that erosion is a complex interaction involving the
effect of the hydrogen ions as well as the effect of the chelating agent. A typical
example of this complex interaction is citric acid. As citric acid dissolves in water, it
dissociates into a mixture of hydrogen ions, acid anion (citrate) and non-dissociated
acid. Citric acid has the capability of producing three hydrogen ions from each
molecule:
HOOCCH2COH(COOH)CH2COOH ↔ HOOCCH2COH(COOH)CH2COO-
+ H+ (3.2)
HOOCCH2COH(COOH)CH2COO-
↔ -OOCCH2COH(COOH)CH2COO
- + H
+ (3.3)
-OOCCH2COH(COOH)CH2COO
- ↔
-OOCCH2COH(COO
-)CH2COO
- +H
+ (3.4)
Citric acid has three pKa values (pKa1= 3.13, pKa2 = 4.76 and pKa3= 6.40). Therefore
citric acid can be found in solution in any of the forms showed in the equations
above depending on the solution pH (Lussi, 2006).
On one side the hydrogen ion can interact with the enamel surface crystals and
combine with phosphate and/or carbonate ions, while on the other hand the chelating
agent (citrate) has high affinity to attract calcium ions as illustrated in Figure 3.3.
FIGURE 3.3 Schematics of citrate ion where two and three of the hydrogen ions have been
lost (a and b respectively) and calcium ion is attracted (Lussi, 2006)
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3.2.3 Prevalence of dental erosion
The prevalence of dental erosion is not well documented since national dental
surveys are not commonly conducted worldwide and rarely include measures of
erosive tooth wear. In addition, it is often difficult to compare the outcomes of
different epidemiological studies on dental erosion due to the use of different
examination standards, including scoring systems, samples and groups examined
(Lussi, 2006). There is however some evidence that the prevalence of erosion is
increasing (Linnett and Seow, 2001, Nunn et al., 2003).
In 1993 the UK National Child Dental Health Survey (Nunn et al., 2003)
included an assessment of the prevalence of erosion of both primary and permanent
incisor teeth. The survey reported that 52% of 5 year old children had erosion on the
palatal surface of their primary incisors with 24% advanced approaching the pulp.
On the other hand the prevalence of erosion on the palatal surface of permanent
incisor was 27% of 15 years old children with 2% showed progression into the pulp
(Lussi, 2006). Studies have shown that socio-economic status may also play a role in
the prevalence of erosion, which could be due to different eating, drinking and
possibly oral hygiene habits. Some studies reported more erosion in higher socio-
economic classes other studies have reported different results, so the issue is still
controversial (Millward et al., 1994, Al-Dlaigan et al., 2001).
At the present time it is clear that dental erosion is an important condition
affecting the dental hard tissues. But there is no clear answer to whether this problem
is actually increasing or whether it has remained constant with figures reflecting only
an increased awareness of the condition.
PART I: INTRODUCTION AND LITERATURE REVIEW
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3.2.4 Methods of dental erosion detection and assessments
Enamel erosion at its early stages is detected as loss of surface contour with a
shiny, glass like appearance which can easily go unnoticed by the patient and/or the
dentist. This is followed by a stage of tooth sensitivity and fracture of thinned
enamel, particularly thinned incisal edges. As erosion progresses more of the
yellowish dentin layer becomes exposed (Figure 3.4).
FIGURE 3.4 Dental erosion affecting both maxillary and mandibular teeth
particularly palatal and lingual surfaces (Lazarchik and Filler, 1997)
Eroded lesions classically look dished out, hard and smooth (Lazarchik and
Filler, 1997). Eccles and Jenkins proposed a set of diagnostic criteria to classify
erosion based on its clinical appearance (Table 3.1).
TABLE 3.1 Eccles and Jenkins erosion grading scale (Lazarchik and Filler, 1997)
Rating Erosion Severity
Grade 0 No involvement of surface
Grade 1 Loss of enamel surface features; no dentin involvement
Grade 2 Exposure of dentin on less than 1/3 of surface
Grade 3 Exposure of dentin on more than 1/3 of surface
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There are several other classifications used in the literature. Some are only
applicable for adults and not children, such as the Smith and Knight Tooth Wear
Index (Smith and Knight, 1984). A modified version of the Smith and Knight Tooth
Wear Index that can be used for children was developed by O’Sullivan et al. (1998).
It is a more detailed index that takes into consideration the site, severity and area
affected. A third index, considered more simple and practical was proposed by Aine
et al. (1993). This index is mainly used for children with gastro-oesophageal reflux
but is suitable for adults and children, primary, mixed and permanent dentition. The
number of different indices for dental erosion indicates that there is no single index
fulfilling all the relevant required criteria. This complicates comparisons between
data obtained from different studies.
3.3 Laboratory techniques for assessment of dental hard tissue
loss
There are many techniques to assess the loss of dental hard tissue and the
softness of the enamel surface. With all the available literature it is now clear that the
complex mechanism of dental enamel mineral dissolution might not be fully
understood and evaluated by a single technique, but instead would require many
techniques with different approaches for full understanding. This section will briefly
mention some of the commonly used techniques.
3.3.1 Scanning electron microscopy
Scanning electron microscopy (SEM) is a qualitative measure. It can be used
to image the surface changes after erosive attacks. It can be used on both polished
and unpolished surfaces after gold sputtering. In enamel, acid attacks due to
PART I: INTRODUCTION AND LITERATURE REVIEW
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immersion of specimens in erosive solutions lead to a surface etching and exposure
of enamel prisms to various extents. For SEM, sample preparation would involve
drying of the specimen which may cause additional alteration to the eroded surface.
Precipitates formed by dissolved enamel minerals may block some enamel surface
and SEM might not detect the blocked enamel prisms in such cases.
3.3.2 Environmental scanning electron microscopy (ESEM)
The ESEM has an advantage over the SEM in that it does not require sample
preparation, and sample examination can be performed without metal or carbon
coating, which reduces the artefacts. Both SEM and ESEM are suitable for use with
native surfaces yet both methods provide qualitative assessment and do not provide
detailed quantitative information about the eroded surface.
3.3.3 Atomic force microscopy (AFM)
Atomic force microscopy (AFM) also provides qualitative measures. The
main application of the AFM is high resolution imaging of different materials. AFM
enables imaging of surface topography as well as differences in elasticity. AFM was
used in many studies for qualitative evaluation of eroded surfaces. It can also be used
to quantitatively measure hardness changes.
3.3.4 Surface profilometry
Surface profilometry involves scanning specimens with a light beam or a
contact stylus with diameter of about 2-20 µm. The contact stylus is loaded with a
force of a few milliNewtons. With surface profilometry complete surface mapping
can be achieved. In cases involving thin and weak enamel surfaces, profilometry
might be affected by the tendency of the contact stylus to penetrate this fragile layer.
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The laser or white light beam stylus has the advantage of having a higher resolution
over the contact stylus and of course beams will not penetrate a fragile surface. Yet it
has the disadvantage of producing over shots at sharp edges such as at the bottom of
a groove and these will result in artefacts.
3.3.5 Nanoindentation and microindentation
The nanoindentation technique is used to investigate enamel dissolution by
measuring the hardness of the enamel surface. It is known that enamel dissolution
involves softening of the enamel surface; therefore the surface hardness
measurement would represent an indirect method in measuring the degree of erosion
or dissolution. Mostly the indenter is a diamond tip which is pressed onto a surface
with a given load and duration, resulting in three sided pyramidal indentation.
Microindentations in sound enamel have typical indentation depths of micrometers
or tens of micrometers, while on the other hand, nanoindentations in sound enamel
have sub-micrometer indentation depths, typically hundreds of nanometres. We
should not forget the fact that the hardness of the surface measured is affected by
many factors like the immediate surrounding material, and material as far away as
ten times the diameter of the indentation itself.
3.3.6 Chemical analysis
Chemical analysis methods are based on the principle that dental enamel
consists of 34%-39% calcium (dry weight) and 16%-18% phosphorus (Lussi, 2006).
Measuring the amount of calcium and/or phosphate dissolved in any solution in
which a dental structure has been placed for some time, gives an indirect estimate of
the amount of demineralisation that has occurred. A calcium sensitive electrode and
PART I: INTRODUCTION AND LITERATURE REVIEW
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a specific pH for the surrounding environment are required for this technique to
work precisely.
Chemical analysis is considered the main competing technique for measuring
mineral loss. It has the advantages of being much cheaper than X-ray based
techniques and its small size makes it easy to carry out in any laboratory. The
chemical method also has the advantage of being able to detect very small mineral
loss using unpolished uncoated native tooth samples, yet these methods are applied
in vitro only (Barbour, 2002).
However it is important to remember that dental enamel dissolution involves
the formation of other phases of calcium phosphate complexes and does not simply
dissolve to its basic constituents of calcium and phosphate. Therefor the
measurement of calcium and/or phosphate in the demineralisation solution may not
be an accurate representative of the amount of demineralisation that took place in the
dental hard structure. Also an intensive solution preparation is required to allow the
measurement of calcium and phosphate with a minimal amount of solution no less
than 100µl.
3.3.7 Microradiography
Microradiography is a method of special interest to this thesis as it is the
technique to be used in all the experiments in this thesis. Therefore, it is discussed in
details in Chapter 9.
The selection of SMR as the technique of choice for the experimental work in
this thesis was based on that SMR was initially developed by Jim Elliott in QMUL
around 1980 and modified by JIM Elliott and Paul Anderson around 1985 giving the
Dental Physical Science Department at QMUL a worldwide reputation in SMR
technology with pioneers working in this field.
PART I: INTRODUCTION AND LITERATURE REVIEW
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CHAPTER 4
Calcium Apatites Dissolution Models
4.1 Introduction
There have been many proposed dissolution models for HAp dissolution
(Dorozhkin, 2002). Each of these models has its own strengths, weaknesses and
limitations. These models provide important information with regards to factors
affecting HAp dissolution. These factors can be classified into:
I: Factors associated with solutions such as pH, composition, saturation, and
hydrodynamics
II: Factors associated with bulk solid such as chemical composition,
solubility and particle size
III: Factors associated with the surface such as defects, absorbed ions, and
phase transformation
In this chapter some of the previously published models for calcium apatite
dissolution models will be discussed in an attempt to highlight the part of the
dissolution mechanism that each model focuses on.
4.1.1 Diffusion controlled and surface controlled models
These types of models are concerned with the study of the dissolution
reaction controlling step, and the transport rates of chemical reagents (H+
and
anions of acids) from solution to the HAp crystal surface and the transport of the
PART I: INTRODUCTION AND LITERATURE REVIEW
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dissolution products away from the HAp crystal surface to the bulk solution
(Ca2+
and PO43-
). Both mechanisms are concerned with the rate controlling
mechanism (driving force), which is the concentration gradient with in the Nernst
diffusion layer in the case of the diffusion model or the gradient of the ionic
chemical potential at the apatite-solution interface in the case of the surface
controlled model (Margolis, 1992).
The question of whether enamel dissolution is a surface or diffusion
controlled or a combination of both is a question that still has no single defined
answer. Some early studies such as those by White and Nancollas (1977) and
Higuchi et al. (1965) described the dissolution of HAp as a diffusion controlled.
Other more recent studies suggest that the dissolution of HAp is not limited purely
by diffusion and that surface processes play an important role in controlling the
overall kinetics depending on the surrounding conditions (Budz and Nancollas,
1988, Anderson et al., 2004).
4.1.2 Self-inhibition (calcium rich layer formation) model
This model was created following studies of the dissolution kinetics of
apatite powders in acidic buffer with solution pH between 3.7 and 6.9, under
constant composition (Dorozhkin, 2002, Tang et al., 2003). It was noticed that
during the initial period of dissolution (first 2-5 min) the amount of Ca2+
released
into the bulk of solution was less than the uptake of H+. This was explained as
follows: as the first amount of Ca2+
is released into the solution some Ca
2+ ions
probably through coupled diffusion are returned from the solution back to the
apatite and adsorb to its surface. This Ca2+
rich surface layer acts as a
semipermeable ionic membrane (Dorozhkin, 1997b).
PART I: INTRODUCTION AND LITERATURE REVIEW
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As the dissolution process continues, more Ca2+
released into the solution
increases, and therefore H+ uptake decreases until electric neutrality is achieved.
Therefore, the overall apatite dissolution process decreases with time (Thomann et
al., 1990, Mafe et al., 1992).
4.1.3 Stoichiometric/Non-stoichiometric dissolution models
Stoichiometric dissolution is also called congruent dissolution; it is the type
of dissolution that occurs when the ions present in the solid dissolve simultaneously
with dissolution rates proportional to their molar concentrations in the solid
(Dorozhkin, 2002). Non stoichiometric dissolution (incongruent dissolution) occurs
when the ions present in the solid dissolve with different dissolution rates from their
molar concentrations (Dorozhkin, 2002), resulting in a situation where a surface
layer is formed with a chemical composition different from that of the bulk of the
solid. It has been reported that in calcium phosphate apatite with a calcium to
phosphate ratio between 1.67 to 2, the calcium ions are the first to dissolve while
when the calcium to phosphate ratio is less than 1.67, the phosphate ions tend to be
the first ions to dissolve. Studies have shown that stoichiometric and non-
stoichiometric dissolution of apatite can occur at the same apatite crystal at different
stages of dissolution, and that whether the apatite will dissolve stoichiometrically or
non-stoichiometrically depends on its chemical composition (Margolis, 1992, Pearce
et al., 1995).
4.1.4 Chemical model
The chemical dissolution model for dissolution of HAp was introduced with
the concept that HAp unit cell (Ca10(PO4)6(OH)2) is unlikely to dissolve by
detachment of a single molecule at a time, breaking down to its 18 ionic components.
PART I: INTRODUCTION AND LITERATURE REVIEW
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Instead, it is expected that HAp would dissolve via a series of chemical reactions
(Dorozhkin, 1997b, Dorozhkin, 1997a)
Previously, the chemical equation for HAp dissolution was thought to be:
Ca10(PO4)6(F,OH)2 + 14H+ → 10Ca
2+ + 6H2PO4
- + 2HF , 2H2O (4.1)
or
Ca10(PO4)6(F,OH)2 → 10Ca2+
+ 6PO43-
+ 2F- , 2OH
- (4.2)
The new concept of HAp dissolution is that apatite would pass through four stages of
chemical reactions to dissolve (Dorozhkin, 2002).
Ca5(PO4)3(F,OH) + H2O + H+ → Ca5 (PO4 )3(H2O)
+ + HF , H2O (4.3)
2Ca5 (PO4 )3(H2O)+ → 3Ca3 (PO4)2 + Ca
2+ + 2H2O (4.4)
Ca3 (PO4)2 + 2 H+ → Ca
2+ + 2CaHPO4 (4.5)
CaHPO4 + H+ → Ca
+2 + H2PO4 (4.6)
During the stages of the dissolution process, different calcium phosphates and
biological apatites can be formed with various stoichiometries which control the
dissolution process by either facilitating or inhibiting it according to the type of
compound being formed.
4.1.5 Nanoscale enamel dissolution model
Traditional understanding of the dissolution process assumes that the
dissolution of minerals is spontaneous and continuous and that all the solid phase can
be dissolved in under saturated solutions until equilibrium is reached. Wang has
lately introduced another vision for the dissolution process (Wang et al., 2005, Wang
et al., 2006) in which the reaction is accompanied by the formation of dissolution
pits and subsequent displacement of pit steps. Pit formation increases surface
PART I: INTRODUCTION AND LITERATURE REVIEW
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roughness. This roughness leads to an increase in the crystal/solution interfacial area.
Subsequent dissolution proceeds through the growth of these pits. However, it has
been found that demineralisation reactions actually involve particle size dependent
critical conditions of energetic control at the molecular level. Only when the pits are
larger than a critical size do they contribute to the reaction, this critical value is of a
nanoscale level. This model of dissolution establishes a clear link between the
microscopic physics of step dynamics and the bulk behaviour of the crystals during
dissolution. It also emphasises the importance of surface energy during dissolution.
4.2 Summary
This brief discussion of the different available models for the study of apatite
dissolution, shows that a complete understanding of HAp demineralisation cannot be
achieved using a single model and whether the model is concerned with the
dissolution process at the solid solution interface, at the solid itself or at the bulk
solution external to the dissolving solid. They all explain HAp demineralisation at
different sites of the HAp that might be taking place simultaneously and are
complementary to each other.
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CHAPTER 5
Zinc
5.1 Introduction
Zinc (Zn2+
) is a metallic chemical element with an atomic number 30. It has
atomic weight 65.39. Its pure metal has a hexagonal close-packed crystal structure.
Its melting point is 420ºC and boiling point 907ºC. Its only common oxidation state
is 2+.
Zinc is found abundantly in tissues throughout the body. Approximately 60%
of total zinc pool is found in muscle tissues, ≈ 30% in bone, ≈ 5% in skin and as a
trace element in teeth (section 2.4) (Christianson, 1991, Hambidge, 2000). It is
involved in many body functions; it is necessary for normal collagen synthesis,
mineralisation of bone, immune system function and proper healing (Thomas and
Bishop, 2007) Therefore, it is considered a dietary essential trace element. It can be
naturally present in some food such as oysters, lobster, most sea food, red meat,
beans and nuts. It is also added to other foods such as cereals and is available as a
dietary supplement (Lawler and Klevay, 1984, Hambidge, 2000, Brooks et al.,
2005). In addition to standard tablets and capsules, some zinc is added to lozenges
and nasal sprays for treatment of the common cold (Weismann et al., 1990, McElroy
and Miller, 2002).
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The current Recommended Dietary Allowances (RDAs) for zinc are 8
mg/day for a female adult and 11 mg/day for a male adult. For pregnant and lactating
women, the RDAs increase up to 12-14 mg/day. The upper margin for the daily
intake of zinc should not exceed 40 mg/day (Maret and Sandstead, 2006). Iron
supplements might interfere with zinc absorption, therefore taking iron supplements
between meals helps reducing their effect on zinc absorption. On the other hand high
zinc intake can inhibit copper absorption sometimes causing copper deficiency and
associated anaemia (Lawler and Klevay, 1984, Milne et al., 1984). For this reason
dietary supplements containing high level of zinc sometimes contain copper as well.
Zinc deficiency is characterised by growth retardation and reduced bone
density as zinc stimulates both bone growth and mineralisation as well as regulating
osteoclast activities (Yamaguchi et al., 1987, Kishi and Yamaguchi, 1994,
Yamaguchi, 1998). Other symptoms include loss of appetite and impaired immune
defense. In more severe cases, zinc deficiency, can cause weight loss, taste
abnormalities, mental lethargy and delayed wound healing. Hair loss, diarrhoea,
delayed sexual maturation, impotence, hypogonadism in males, eye and skin lesions
are also not uncommon (Maret and Sandstead, 2006) in severely zinc-deficient
patients.
The difficulty of diagnosing zinc deficiency lies in that none of these
symptoms is specific and they are often associated with other health conditions.
Therefore, a medical examination is necessary to diagnose zinc deficiency (Golden,
1989). Zinc ion levels in the body are difficult to measure using laboratory tests,
because of their distribution throughout the body as a component of many proteins
and nucleic acids. Plasma and serum zinc level are the most commonly used for
testing zinc deficiency. People with gastrointestinal diseases such as Crohn’s disease
PART I: INTRODUCTION AND LITERATURE REVIEW
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and ulcerative colitis are more susceptible to zinc deficiency as gastrointestinal
diseases may increase the loss of zinc from the gastrointestinal tract and lower zinc
absorption or uptake (Wapnir, 2000).
Zinc toxicity can occur in both acute and chronic forms. Acute adverse
effects of high zinc intake include nausea, vomiting, loss of appetite, abdominal
cramps, diarrhoea, and headache. Approximately 500 mg zinc can cause acute
toxicity while the intake of 150-450 mg zinc per day is enough to cause chronic
toxicity (Fosmire, 1990).
5.2 Zinc in the oral cavity
Zinc is naturally present in the oral cavity, in the teeth, saliva and dental
plaque. It is one of the trace elements present in teeth and shows a distribution
pattern similar to that of fluoride and lead (Robinson et al., 1995a) with higher
concentration at the surface structure of dental enamel and lower concentrations at
the subsurface. Concentrations of zinc in the subsurface enamel of teeth range from
430 to 2100 parts per million (ppm), with most zinc deposition taking place before
tooth eruption (Brudevold et al., 1963, Brudevold et al., 1975). After eruption, zinc
concentration at the enamel surface increases further, suggesting incorporation
occurring during post eruption exposure to oral fluids. With ageing excessive zinc
content is lost over the years in a similar fashion to fluoride (Weatherell et al.,
1972, Weatherell et al., 1973).
Zinc concentration analysis through cross sections of the tooth crown show
highest zinc concentration in the enamel surface layer and decrease in concentrations
towards the dentino-enamel junction. In dentine there is also a gradient in zinc level
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with the greatest concentration occurring adjacent to the pulp. The level of zinc in
the bulk of the coronal dentine is approximately the same as that in junctional
enamel. Near the pulp zinc concentrations increase sharply and approach those of
external enamel (Brudevold et al., 1963).
Much research has been conducted to investigate zinc concentrations in
saliva. A range of values between 0.01 to 0.2 ppm have been reported (Bales et al.,
1990, Oezdemir et al., 1998, Watanabe et al., 2005, Burguera-Pascu et al., 2007).
Zinc is also naturally present in dental plaque and researchers have studied zinc
concentrations in both dry as well as wet dental plaque. It was found that zinc
concentrations in dry plaque ranged between 6 ppm and 31 ppm, which is estimated
to be around seven folds more than the reported zinc concentration in wet plaque.
The difference in concentrations between the dry and wet plaque is justifiable
assuming that drying increases the apparent concentration (Tatevossian, 1978, Agus
et al., 1980, Duckworth et al., 1987).
5.3 Effect of zinc on calculus formation
5.3.1 Zinc containing mouthwashes
Mouthwashes containing zinc salts were first reported to reduce dental plaque
growth in the early 1970s (Picozzi et al., 1972, Fischman et al., 1973), followed by
other studies investigating the effect of zinc containing mouthwashes on dental
plaque growth, and calculus formation (Schmid et al., 1974, Compton and Beagrie,
1975, Skjörland et al., 1978),
The role of zinc in calculus formation was confirmed in later work (Harrap et
al., 1983) which stressed the importance of the use of high concentrations of zinc
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and sufficient frequency of application to suppress calculus formation (Harrap et al.,
1984). Prolonged retention of zinc in the mouth is thought to be important for its
activity (Bonesvoll and Gjermo, 1978, Afseth et al., 1983a)
After using mouthwashes containing zinc salts, approximately 40% of the
amount of the applied zinc is retained in the oral cavity. Its concentration rapidly
decreases to a low concentration yet significantly above the zinc baseline in 30 to 60
min. This rapid clearance phase is followed by a slow clearance phase that extends
for many hours. The elevated zinc concentration persists in dental plaque for up to
13 hours (h) after application. The incorporation of zinc citrate to mouthwashes was
reported to successfully reduce plaque by approximately 8% (Addy et al., 1980), but
the clinical significance is unknown.
5.3.2 Zinc containing toothpastes
Toothpastes are more widely used than mouthwashes. Therefore they are
considered a more desirable method for delivering an antiplaque agent. Yet the
incorporation of antiplaque ingredients into toothpastes presents several difficulties.
Toothpastes formulations are quite complex and some of the ingredients may affect
activity of the therapeutic agent. For example the availability of chlorhexidine is
reported to be affected by anionic detergents usually present in toothpastes (Addy et
al., 1992). Also the concentrations of the antiplaque ingredients should be higher
than in mouthwashes as the dose of dentifrice used in the mouth is only about 0.1 to
0.2 of that used in the mouthwashes.
Zinc was introduced into toothpastes in the form of zinc citrate. Literature
review shows much research done on this. Studies have managed to clearly show
that zinc containing toothpastes show the same antiplaque activity as that reported
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for zinc in mouthwashes (Fischman et al., 1973, Schmid et al., 1974, Skjörland et
al., 1978, Harrap et al., 1983, Harrap et al., 1984).
The mechanism by which zinc affects plaque growth is not clearly
established. Zinc might bind to the oral bacterial surface altering its surface potential
(Ollsenn and Glantz, 1977) and accordingly might affect bacterial adhesion to teeth
(Skjörland et al., 1978). Or, it might be zinc’s capability to inhibit acid production by
bacteria in plaque (Oppermann and Rölla, 1980, Oppermann et al., 1980, Harrap et
al., 1983) by altering the metabolic activity of the oral bacteria hence reducing
bacterial growth (Afseth, 1983, Afseth et al., 1983c, Saxton et al., 1986, Hall et al.,
2003).
Zinc has been used for a long time for its antiplaque activity as well as to
reduce oral malodor. Oral malodor (halitosis) is a condition that originates from
bacterial metabolism of proteins from saliva, sloughed oral tissue and food debris
leading to the formation of amines, alcohol and particularly volatile sulphur
compounds such as hydrogen sulfide (H2S) (Young et al., 2001, Young et al., 2003).
Zinc salts are found to be highly effective in reducing H2S since they are chemically
able to neutralise H2S as well as acting as antimicrobial agents (Bradshaw et al.,
1993).
Oral availability in adequate quantities is a necessary prerequisite of any
agent for antiplaque activity in vivo. Data demonstrates that approximately 30% of
zinc citrate is retained in the oral cavity after brushing (Cummins, 1991). Gilbert and
Ingramm (1988) had demonstrated that after brushing with 1gm toothpaste
containing zinc, 25 to 38% of the zinc was retained in the oral tissues. Zinc levels in
saliva remained significantly above baseline level for at least 2 h after application.
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Another study determined a significant increase in salivary zinc levels,
highest 5 minutes after brushing with toothpaste containing 0.75% zinc citrate. This
was followed up by gradual reduction in zinc concentration, approximately reaching
the base line levels after 7 h (Oezdemir et al., 1998).
5.4 Effect of zinc on dental caries
Due to the success of incorporating zinc in toothpastes and mouthwashes and
the demonstration of their ability to reduce plaque and calculus, zinc containing
toothpastes and mouthwashes have been used in treating and preventing periodontal
diseases (Mellberg and Chomicki, 1983).
Since zinc incorporation in toothpastes has extended to involve its
incorporation in some fluoridated toothpastes, more research work was needed to
determine if zinc might affect fluoride deposition in dental enamel and whether its
incorporation in fluoridated toothpastes showed a synergistic/ antagonist or no effect.
Mellberg and Chomicki (1983) suggested that zinc citrate inhibits fluoride
uptake by artificial enamel caries and gave two explanations: either the inhibition is
due to zinc reaction with monofluorophosphate (MFP) ions in the solution inhibiting
its reaction with enamel, or most likely there is reaction of zinc with the phosphate
ions in the enamel lesion (caries) which leads to the formation of insoluble zinc
phosphate complex. Zinc phosphate complex coats the HAp surface, precipitates and
blocks the diffusion of fluoride into the carious sites.
On the other hand more recent in vivo studies have demonstrated a reduction
in enamel demineralisation with the use of zinc containing fluoride toothpastes
(Lynch, 2011). However the demineralisation reduction could not be entirely due to
the interaction of zinc with HAp as it may, to a degree, be the result of the
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antimicrobial effect of zinc (Ten Cate, 1993, Churchley et al., 2011). Further
research is recommended to study the direct and individual effect of Zn2+
on enamel
demineralisation.
5.5 Effect of zinc on dental erosion
As mentioned before, most of the research done on zinc has concentrated on
zinc effects on dental plaque and calculus formation which is indirectly linked to,
management of oral malodor, and to a lesser extent on zinc anti-caries effects.
A review of the literature on the effect of zinc on dental erosion did not
reveal any work done on the use of zinc as a preventive aspect or in cases of dental
erosion. In fact many publications have studied the erosive potential of zinc fumes
(zinc oxide, zinc chloride) specially on industrial workers (Remun et al., 1982).
Zinc’s ability to inhibit apatite dissolution under acidic erosive like
conditions and the potential usefulness of zinc as an ingredient in toothpastes for
erosion prevention purposes is a subject that has been overlooked and requires
further research.
5.6 Effect of zinc on hydroxyapatite dissolution
The exact mechanism by which the divalent cations reduced enamel
dissolution has been an issue of controversy as ion uptake by HAp from solution can
occur via two methods.
Method 1: As HAp dissolves in the acidic environment, phosphates are released.
Phosphates can react with metal cations in the solution to form new low soluble
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divalent metal (Me) phosphate crystals with an apatitic structure that precipitates
according to the Equation 5.1 and 5.2:
Ca10(PO4)6(OH)2 + 14H+ → 10Ca2
+ + 6H2PO4
- + 2H2O (5.1)
10Me2+ + 6H2PO4
- + 2H2O → Me10(PO4)6(OH)2 + 14H
+ (5.2)
Method 2: This involves some Ca2+
being substituted with the divalent metal cation
by a diffusion process and adsorbed onto the surface (Equation 5.3)
Ca10(PO4)6(OH)2 + xMe2+
→ (Ca10-x)Mex(PO4)6(OH)2 + xCa2+
(5.3)
Therefore, we can say that zinc might adsorb on to the HAp surface and block
high energy “kink” sites on the outer surface. According to Xu et al. the adsorption
method occurs at pre equilibrated HAp. Otherwise zinc might be incorporated in the
HAp lattice forming new zinc phosphate crystals that precipitate (Xu et al., 1994). At
zinc concentrations of ≥1ppm, hopeite (Zn3(PO4)2.4H2O) is formed. Zinc is
incorporated into the HAp lattice forming a hopeite layer at the surface (Xu et al.,
1994). Hopeite is usually formed at low pH. As the pH increases, other forms of
apatitic structures such as scholzite (CaZn2(PO4)2.2H2O) and zincite (ZnO) are
formed.
The incorporation of zinc as a divalent metal cation in HAp and in particular
its binding site is still not clearly understood. One reason for this uncertainty is the
presence of two structurally distinct cation sites Ca1 and Ca2, in the HAp lattice
which appear to be suitable for zinc substitution (Figure 5.1). A considerable amount
of research has been done on metal ion preference in the HAp structure (Mayer et
al., 1994, Terra et al., 2002, Tamm and Peld, 2006, Matsunaga, 2008, Tang et al.,
2009, Matsunaga et al., 2010) and still the debate continues regarding the selection
criteria influencing how metal ions choose between Ca1 and Ca2 sites.
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When Zn2+
occupies the Ca2 site, the result is an overall shrinkage and more
stability in the crystal. The local HAp lattice shrinkage brings the ZnO4 tetrahedron
and the channel OH- groups in the HAp lattice closer, minimising the effect on the
adjacent Ca1 sites and avoiding any disruption of the framework (Elliott, 1994).
FIGURE 5.1 Schematic figure for the structure of Zn-doped HAp, where yellow,
blue, red, black, green and gray refer to calcium1 site,calcium2 site,oxygen,
hydrogen, zinc and phosphate groups respectively (Tang et al., 2009)
Ca2 site preference is in case of pure HAp, but in biological apatite when
there is an especially high concentration of carbonate (CO32-
) and which also may be
Ca2+
deficient which is the case in teeth, this might influence the uptake of Zn2+
and
its site binding. From reviewing the literature and the mechanism through which
Zn2+
affects the hydroxyapatite demineralisation rate (RDHAp) it seems that both,
adsorption and incorporation are not mutually exclusive, and it is likely that both
mechanisms are implicated in reducing HAp solubility in the presence of Zn2+
, to a
greater or lesser extent.
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CHAPTER 6
Strontium
6.1 Introduction:
Strontium (Sr) is one of the most abundant elements on earth, forming about
0.04% of the earth’s crust. It is element number 38 of the periodic table of elements,
and was first discovered in 1808 near a village in Scotland called Strontian, after
which the metal was named (Murray, 1993). It has mass number 87.62, a melting
point of 777°C and a boiling point of 1384°C. Strontium can exist in two oxidation
states: Sr+ and Sr
2+. Under normal environmental conditions, only the Sr
2+oxidation
state is stable enough to be important. Strontium is reactive with water to produce
strontium hydroxide and hydrogen gas. Natural strontium is not radioactive and
exists in four stable types (or isotopes), each of which can be written as 84
Sr, 86
Sr,
87Sr, and
88Sr. Rocks, soil, dust, coal, oil, surface and underground water, air, plants,
and animals all contain varying amounts of strontium. Strontium concentrations in
most materials are a few ppm, yet strontium is considered abundant trace element in
seawater, at an average concentration of 8.1 ppm (Angino et al., 1966). The human
body contains an average of 320 mg of strontium, almost all of it is in bone, teeth
and connective tissue (Schweissing and Grupe, 2003).
Strontium compounds, such as strontium carbonate, are used in making
ceramics and glass products, paint, fluorescent lights, medicines, and other products.
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Strontium can also exist as radioactive isotopes. 90
Sr, or strontium ninety, is
the most hazardous of the radioactive isotopes of the chemical element strontium.
90Sr is formed in nuclear reactors or during the explosion of nuclear weapons. The
radioactive half-life is the time that it takes for half of a radioactive strontium isotope
to give off its radiation and change into a different element. 90
Sr has a half-life of 29
years.
Strontium is not an essential trace element, and therefore, there is no
established recommended daily intake, no defined level of deficiency and no
identified symptoms of strontium toxicity or strontium overdose. It is usually
abundant in milk, dairy products, vegetables (such as spinach, lettuce, and carrots),
red meat as well as seafood. Therefore the body usually gets the little strontium it
needs through diet. However, therapeutic doses of strontium supplements range from
10 mg to 1000 mg and more daily. Such a high dose is usually prescribed for the
treatment of osteoporosis, as strontium plays a role in promoting osteoblastic, and
inhibiting osteoclastic, activity (Meunier et al., 2004).
Once strontium enters the bloodstream, it is distributed throughout the body,
where it can enter and leave the cells quite easily. In the body, strontium behaves
very much like calcium. Most of the strontium will accumulate mainly in bone (in
adults, strontium mostly attaches to the surfaces of bones). Strontium is eliminated
from the body through urine, faeces, and sweat.
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6.2 Strontium in bone
Strontium has close chemical similarity to calcium; therefore it behaves in a
similar manner to calcium and is involved in the development of tooth and bone at
times when calcification is taking place. Strontium can replace calcium to some
extent in various situations in the body, such as replacing a proportion of calcium in
the hydroxyapatite lattice in bone and teeth.
The human placenta plays a selective role against strontium transfer from
maternal blood to the foetus during early pregnancy; this selective permeability
becomes free passage towards the end of pregnancy. The strontium concentration of
the foetus is determined entirely by the strontium level in the mother’s blood
(ingested by the mother during pregnancy).
According to very early studies, strontium deposition in bone can take place
through two methods (Likins et al., MacDonald et al., 1951, Glas and Lagergren,
1961).
Method one: involves rapid incorporation of strontium. It refers to the blood
strontium deposited by ionic exchange, surface adsorption, and preosseous protein
binding.
Method two: involves slow incorporation of strontium into the lattice structure of the
bone crystals during their formation.
Method one and method two are both considered valid and we cannot be certain
about which of the two strontium deposition processes contributes to the initially
formed bone and tooth tissues.
Most recent studies have shown that postnatal and through life, strontium
accumulates in bone, in particular where active remodeling is taking place as it
stimulates the cell replication of osteoblasts which ultimately increase the rate of
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new bone formation and decrease bone resorption by inhibiting osteoclast
differentiation and activity (Canalis et al., 1996, Marie et al., 2001, Baron and
Tsouderos, 2002). Accordingly strontium has been used in medications for the
treatment and prevention of osteoporosis (Bonnelye et al., 2008).
6.3 Strontium in the oral cavity
Strontium in the oral cavity is present in teeth, dental plaque as well as in
saliva. There has been considerable research on the role and distribution of trace
elements in dental enamel, and these have succeeded in demonstrating the
concentration distribution pattern through the dental enamel thickness. While most
of the trace elements studied (eg. Zn2+
, Cu2+
, F-, Fe
2+, Mn
2+) showed a higher
concentration at the outer enamel layer, Sr2+
and Mg2+
showed a different
distribution pattern. Their concentration gradually increased with age and more
towards the dentino-enamel junction (Frank et al., 1989, Reitznerová et al., 2000).
Human enamel was reported to have mean values between 70 and 286 µg/g of
strontium, with a median value of 115 µg/g (Curzon and Cutress, 1983). Less
strontium is found in dentine than in enamel (Frostell et al., 1977, Frank et al.,
1989). Strontium concentrations on tooth surfaces can be affected by the amount of
strontium in the drinking water. The issue of the relationship between Sr2+
concentration in water and in the enamel surface and its relation to caries resistance
has been a topic of interest since the 1950s (Steadman et al., 1958, Barmes, 1969).
Although Sr2+
is not considered one of the elements with significant
quantities in dental plaque, it has been detected in plaque fluid from subjects who
lived in an area where the strontium level in drinking water ranged between 0.4 and
17.9 mmol/l (Margolis, 1994).
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Curzon studied the whole resting saliva for 14 year old school children in
different areas with different strontium levels in drinking water in Wisconsin
(U.S.A.) and found that strontium concentrations in saliva were weakly related to its
concentrations in drinking water. He also reported a negative relationship between
strontium concentrations in saliva and caries prevalence (Curzon, 1984).
6.4 Effect of strontium on hydroxyapatite dissolution
The mechanism of Sr2+
behaviour in HAp is controversial. Grynpas (1993)
thought that the incorporation of Sr2+
in the HAp lattice weakened the lattice
structure and increased its solubility. Le Geros (1991) also found that the substitution
of some Ca2+
in calcium apatite by Sr2+
causes the crystal lattice to expand and the
solubility to increase. This is due to the larger ionic radius of Sr2+
(≈1.12Å) than the
ionic radius of Ca2+
(≈0.99Å) (Kikuchi et al., 1994). On the other hand
Christoffersen et al. (1997) and Dedhiya et al. (1973) found that Sr2+
strongly
inhibited HAp dissolution due to the formation of a Ca3Sr2(PO4)3OH surface
complex, with up to 40% strontium substitution (Heslop et al., 2003). It was also
indicated by Christoffersen et al. (1997) that the solubility of strontium-substituted
apatite increases with the increase in strontium content. In comparison with results
obtained from Verbeeck et al. (1981), up to 10% Strontium substituted apatite give a
reduction in HAp dissolution (Li et al., 2007).
6.5 Effect of strontium on dental caries
Research work on the effect of strontium on dental caries goes back to as the
mid-1960s, when Losee and Adkins showed that post eruption exposure to high
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strontium doses had an anti-carious effect. Gedalia and Curzon also studied the
effect of prenatal, pre-eruptive and postnatal administered strontium on rat teeth and
found that strontium showed an anti-carious effect (Gedalia et al., 1975, Joseph et
al., 1977, Ashrafi et al., 1980, Curzon et al., 1982). The pre-and post-eruptive effects
of low doses of strontium on dental caries in rats were reported to be associated with
the lowest caries level. It was also reported that the uptake of strontium by enamel
was significantly correlated with its concentration in diet (Ashrafi et al., 1980).
In 1969 Losee and Adkins (1969) published a 10 year study carried out by
the United States Naval Dental Service which involved dental examination of
approximately 270,000 naval recruits, and showed only 360 completely caries-free
individuals. Out of the 360 caries-free individuals, 36 individuals belonged to one
small area near Rossburg, Ohio, where the water had a higher strontium
concentration. Likewise, Curzon (1985) conducted studies on 80 young boys from
five different communities in Ohio and his results indicated an inverse relationship
between caries prevalence and strontium level in drinking water. Curzon et al.
(1978) also carried out a study on 1313 children aged 12 to 14 years and suggested
that strontium in drinking water supplies may be associated with an inhibition of
dental caries, particularly during the tooth development period, presumably through
incorporation in the apatite crystal. Similar results were obtained from Athanassouli
et al. (1983) who investigated the possible cariostatic effect of high strontium levels
in drinking water and concluded that a low DMFT index was associated with high
strontium concentration in drinking water.
Studies on the effects of strontium and fluoride applied together showed that
the combination appeared to be more effective in controlling dental caries than
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fluoride alone (Featherstone et al., 1983a, Curzon, 1985, Curzon, 1988, Thuy et al.,
2008).
6.6 Effect of strontium on dentine hypersensitivity
Strontium containing toothpastes for the treatment of tooth hypersensitivity
were introduced to the market around five decades ago. Strontium chloride was
introduced commercially as the first tubule occluding agent in the original
Sensodyne™ toothpaste (Dowell and Addy, 1983). Due to the reaction that occurs
between strontium chloride and fluoride, an insoluble strontium fluoride is formed
and that is the rational for calling the original Sensodyne™ product a fluoride free
toothpaste. In the 1970s however, strontium chloride was mostly replaced by
potassium nitrate. Strontium containing toothpastes were later modified by the
incorporation of strontium acetate in place of strontium chloride. Strontium acetate is
compatible with fluoride and does not form insoluble precipitates (Cummins, 2010).
Eight percent strontium acetate showed rapid and lasting relief of hypersensitivity
(Layer and Hughes, 2010). Together 8% strontium acetate with 1040 ppm fluoride
are considered the optimal combination available currently on the market for the
treatment of tooth hypersensitivity (Hughes et al., 2010, Mason et al., 2010). Three
potential mechanisms of action for strontium salts, in terms of treatment for dentine
hypersensitivity have been proposed in the literature. First, it is believed that
strontium causes some degree of nerve depolarisation. Second, strontium shows
chemical similarities to calcium and is capable of replacing lost calcium in the HAp
lattice. Third, a layer of fine particles may be deposited by the strontium salts leading
to the occlusion of the dentinal tubules.
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In conclusion, strontium has proved its effectiveness in the management of
tooth hypersensitivity. However, its anti-carious effect is still an area of controversy
and more research is needed.
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CHAPTER 7
Copper
7.1 Introduction:
Copper (Cu2+
) is a highly conductive metal (thermally and electrically). It has
atomic number 29 and mass 63.546. Its melting point is 1084.62ºC and its boiling
point is 2562ºC. Copper is a transition metal with different oxidation states: Cu1+
(cuprous), Cu2+
(cupric), Cu3+
and Cu4+
. The cupric state is found most often in
biological systems. The name copper originates from the word Cyprium (means
metal of Cyprus) which was later on shortened to Cuprum and this goes back to the
Roman Empire when copper was discovered in Cyprus (Dhavalikar, 1997).
Copper is an essential trace element for human metabolism. It is needed for
many body functions such as red blood cell synthesis, synthesis of particular
enzymes responsible for body metabolism and, energy production, and it also assists
in iron absorption (Danks, 1988). Copper also forms part of the enzyme imine
oxidase which is involved in collagen crosslinking (Knott and Bailey, 1998).
Copper is abundant in regular diets. The RDA of 2 mg is usually obtained
easily from a balanced diet. It is rare to be truly deficient in copper (Klevay, 1998).
Copper is found in seafood, organ meat (such as liver, kidney and heart), nuts (such
as cashew and almond), soybeans, lentils as well as dried fruits (Klevay, 1998).
Humans may also obtain copper inadvertently using copper cookware. When food is
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prepared and left to set for an extended period of time in copper cookware, this may
allow copper transfer from the cookware surface. One may also get copper
unnoticeably from water coming through copper pipes. In many regions of the world,
drinking water supplies are constructed from copper tubing. Copper plumbing
leaches a small amount of copper into drinking tap water supplies. The WHO has
published a document in 2004 about copper in drinking water (WHO, 2004).
Copper deficiency can occur in early life due to insufficient copper in infants
exclusively fed a cow’s milk diet, because of the low copper content of cow’s milk,
and its limited absorption into cow’s milk (Dorner et al., 1989). In adult life, copper
deficiency can arise after burns, chronic diarrhoea, intestinal diseases and pancreatic
diseases.
Acute copper toxicity is very rare and mainly restricted to the accidental
drinking of solutions of copper nitrate or copper sulphate. However, these solutions
and other organic copper salts have a powerful emetics effect and in large doses they
are normally rejected by the body by vomiting. Chronic copper poisoning is also
very rare in healthy humans as healthy human livers are capable of excreting
considerable amount of copper (Turnlund et al., 1990, Turnlund et al., 1998).
7.2 Effect of copper on dental plaque
In 1940, Hanke reported the effect of copper on dental plaque and referred to
this as the “destruction of plaque” (Hanke, 1940). Since then, the antimicrobial effect
of copper ions on oral bacteria has been a subject of interest for researchers, but the
antibacterial effect of ions other than copper, particularly zinc or silver on biofilm
formation, appears to have received much more attention.
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In vitro studies have reported the antibacterial effect of copper ions against
oral bacteria. Due to the variety of oral microorganisms and the variety of tests, the
extent of the effect of copper ions has been different across the various studies, and
therefore difficult to compare. For example, Maltz and Emilson (1982) studied the
effects of various fluoride salts on oral bacteria. They and others reported
bactericidal effects of copper fluoride on several species of oral bacteria, and
concluded that metal salts of fluoride (SnF2 and CuF2) showed a stronger
antibacterial effect than non-metal fluoride compounds, which is in accordance with
other studies (Andres et al., 1974, Yoon and Berry, 1979, Mayhew and Brown,
1981).
In vivo studies have also shown that copper ions have antibacterial activity.
There is a controversy about whether chlorhexidine is more of an efficient
antibacterial agent than copper. Waerhaug et al. (1984) reported that the antibacterial
effect of copper ions was not as noticeable as that of chlorhexidine, which has been
reported to be the most effective antibacterial agent for the reduction of plaque and
gingivitis (Waerhaug et al., 1984, Ciancio, 1992). However, Waler and Rolla (1982)
have studied and compared the effect of chlorhexidine, copper, and silver containing
solutions, and found that although chlorhexidine showed the best results it was not
significantly different from the effects of copper ions, whereas the efficacy of silver
was the least statistically significant. Whether the antimicrobial effect of
chlorhexidine is significantly better than that of copper or not, copper has the
advantage of causing less staining than chlorhexidine which causes darker and more
difficult stains to remove (Mandel, 1988). Also, the taste of both copper and
chlorhexidine mouthwashes is a problem, but copper containing mouthwash is
considered to be more acceptable than chlorhexidine mouthwashes (Waerhaug et al.,
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1984). Thus, copper containing products show promise for future use in the
treatment of oral infections and deserve further study (Mandel, 1988).
7.3 Effect of copper on dental caries
As discussed in Section 7.2, studies have shown that copper salts exhibit an
inhibitory effect on bacterial dental plaque. Studies on the ability of copper to inhibit
dental caries initiation and progression go back as far as the 1950s when it was
reported that copper has an inhibitory effect on dental caries in hamsters (Hein,
1953). Later Afseth et al. studies investigated the cariostatic effect of copper on rats
and on human dental enamel (Afseth et al., 1980, Afseth et al., 1983b, Afseth et al.,
1984a, Afseth et al., 1984b).
Both in vivo and in vitro studies, showed copper as a potent cariostatic agent.
Its cariostatic property is demonstrated through its ability to reduce the number of
bacteria in dental plaque as well as decrease smooth surface dental caries scores
(Oppermann and Johansen, 1980, Afseth et al., 1983b, Mandel, 1988, Davey and
Embery, 1992).
Afseth et al. studied the effects of copper sulphate (in the form of a
mouthwash), fluoride (in the form of fluoridated water), and the combination of
both, on dental caries in rats. They noticed that the group receiving topical Cu2+
treatment together with fluoride in the drinking water gave the lowest smooth surface
caries score and the lowest number of bacteria in dental plaque. These results were
comparable to results found in previous studies (Larson and Amsbaugh, 1975,
Afseth et al., 1984a).
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According to the literature, copper exerts its cariostatic function through two
mechanisms. First, is the antibacterial action of copper on dental plaque bacteria
(bactericidal/bacteriostatic effect). Copper has the ability to a) limit bacterial growth,
by inhibiting glycolysis through oxidation of thiol groups in the enzymes involved in
the glycolysis process, leading to decreased acid production by bacteria, and b)
stopping important metabolic reactions in plaque bacteria such as the bacterial ability
to convert urea to ammonia (Maltz and Emilson, 1982, Afseth et al., 1984b, Rosalen
et al., 1996a, Rosalen et al., 1996b). Second is the ability of copper to form copper
phosphate crystals on the tooth surface that protect the enamel and increase its
resistance to acidic mediated dissolution. However, very few studies have been
carried out to verify this second mechanism (Koulourides et al., 1968, Rosalen et al.,
1996a, Brookes et al., 2003, Abdullah et al., 2006) and this is one of the aims of this
thesis.
7.4 Effect of copper on enamel demineralisation
A literature review of the effects of copper on dental enamel demineralisation
shows that most research has been done to explore its cariostatic effects due to its
bactericidal properties. Only a few studies have been carried out to examine the
direct effect of Cu2+
on the acid mediated dissolution mechanism of dental enamel.
Afseth et al. studied the effect of copper applied topically or in drinking
water on the caries experience in rats. They reported that 1.0 mmol/l Cu2+
in drinking
water and 5.0 mmol/l Cu2+
, topically applied, inhibited caries formation in a rat
model. They also reported that although the Streptococcus mutans count was
lowered when copper was delivered topically or in drinking water, the Streptococcus
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mutans count was only statistically significantly reduced when copper was delivered
in drinking water. This shows that copper may have a direct effect on enamel
demineralisation, which Afseth et al. explained with reference to the ability of Cu2+
to electrostatically bind to various acid groups in dental plaque and stay retained in
dental plaque for a long duration (Afseth et al., 1984a, Afseth et al., 1984b).
Brookes et al. (2003) studied the inhibitory effect of copper in the form of
copper sulphate under erosion-like conditions using acetic acid pH 3.2. They found
that copper decreased enamel dissolution, and by studying a range of copper
concentrations, they found that the peak of the reduction in enamel dissolution was
achieved by 90 ppm Cu2+
, whereas higher copper concentrations did not show a
statistically significant reduction in enamel dissolution rate.
FIGURE 7.1 The effect of Cu2+
concentration on the phosphate released form powdered
human enamel (Brookes et al., 2003) after the conversion of Cu2+
concentrations from
mmol/L to ppm
The same group measured the molar calcium to phosphate ratio in the
demineralisation solution in the presence of Cu2+
, it was found that there is a higher
calcium to phosphate ratio in the demineralisation solution compared to the calcium
0.0
0.2
0.4
0.6
0.8
1.0
0 20 40 60 80 100 120 140 160 180 200
Cu2+ concentration (ppm)
Ph
osp
hate
lo
ss f
rom
en
am
el
po
wd
er
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to phosphate ratio in enamel (≈1.88 compared and ≈1.55, respectively) suggesting
that copper ions replace calcium ions, forming copper phosphate crystals on the
enamel surface. This has a more stable structure which has a lower dissolution rate
when exposed to an acidic attack (Abdullah et al., 2006). They concluded that the
Cu2+
inhibition effect of enamel demineralisation may be a surface controlled
mechanism rather than a change in structural phase (Brookes et al., 2003) and might
even occur at the level of the Stern layer as discussed by Mafe et al. (1992).
In conclusion, this literature review shows that Cu2+
has potential usefulness
as a cariostatic agent both as an antimicrobial agent against dental plaque bacteria
causing caries and periodontal disease, and as a mineral mass loss protective agent,
against caries and erosion. More research is needed to explore the mechanisms by
which Cu2+
alters HAp dissolution kinetics.
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CHAPTER 8
X-ray microscopy
8.1 Nature of electromagnetic radiation
Radiation can be defined as the transmission of energy through space and
matter (White and Pharoah, 2008). This transmission can take place in two forms;
particulate and electromagnetic. Particulate radiation consists of atomic nuclei or
subatomic particles moving in a high velocity such as α-rays and β-rays, while
electromagnetic radiation is the movement of energy through space as a combination
of electric and magnetic fields (White and Pharoah, 2008).
Electromagnetic radiation is a wave in space or through matter with the
electric and magnetic field components perpendicular to each other and
perpendicular to the direction of energy propagation as demonstrated in Figure 8.1
(Seibert, 2004) .
FIGURE 8.1 X-ray is an electromagnetic wave, where the electric and magnetic
fields are perpendicular to each other and to the direction of propagation (Seibert,
2004)
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Electromagnetic radiation is classified into several types according to their
wave frequency. Radio waves, microwaves, infrared radiation, visible light,
ultraviolet radiation, X-ray and gamma rays are all examples of electromagnetic
waves. Of these, radio waves have the longest wave length and gamma rays have the
shortest (Figure 8.2).
FIGURE 8.2 The electromagnetic spectrum in terms of wave length
(http://www.centennialofflight.gov/essay/Dictionary/ELECTROSPECTRUM/DI159.htm)
8.2 X-ray generation
8.2.1 Introduction
More than one hundred years ago, in 1895, Wilhelm Conrad Roentgen
discovered X-ray generation. He was the first to call them X-rays. One of the first X-
ray photographs taken was the hand of Roentgen’s wife taken three days before
Christmas on 22 December 1895 (Figure 8.3). The image displayed both her
wedding ring and bones (Assmus, 1995).
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FIGURE 8.3 First X-ray photograph taken by Roentgen showing his wife’s fingers
(Assmus, 1995)
To generate X-rays Roentgen used a large induction coil connected to
vacuumed glass tube. His detection system comprised of a paper screen covered with
crystals of barium platinocyanide, set up in a dark room. On 28 December 1895 he
announced his discovery and gave an accurate description of many of the basic
properties of the rays (Assmus, 1995).
8.2.2 Modern X-ray tube
Roentgen’s idea of X-ray generation was to introduce a high voltage to a
residual gas at 10-3
mmHg pressure, leading to the formation of electrons and
positively charged ions. The positive ions bombard a curved cathode releasing
electrons which are accelerated towards the anode under high voltage producing X-
ray. In roentgen X-ray generation, it was essential to maintain the gas pressure
constant because changes in the pressure resulted in change in voltage between the
anode and cathode of the tube.
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In 1913 William Coolidge introduced a new source of electrons in the form
of a hot tungsten spiral filament in a vacuumed glass. The filament is heated by a
current provided by a battery and accordingly the electron current could be
controlled independently of the applied voltage (Assmus, 1995).
The basic operating equipment for generating X-rays is the X-ray tube, and is
composed of cathode and anode. The cathode acts as a source of the electrons to be
directed at the anode. Both anode and cathode are enclosed in an evacuated glass
tube. Electrons from the cathode are generated and when they strike the target in the
anode they produce X-rays.
In order for an X-ray tube to generate X-rays it is fundamental that it should
have a power supply that is capable of establishing a high voltage potential between
the anode and the cathode which is required to accelerate the electrons (Figure 8.4).
FIGURE 8.4 Schematic diagram showing basic components of an X-ray tube (a)
and X-ray tube used in SMR machine (PANalytical®
) with silver (Ag) target (b)
(b) (a)
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I. Cathode
The cathode consists of a filament and a focusing cup (Figure 8.4(a)). The
filament is the source of electrons. It is a coil of tungsten wire about 2 mm in
diameter and 1 cm in length, mounted on two stiff wires that act as holder and at the
same time supply the filament with electrical current. To achieve a small focal spot
the electrons are focused by a small metal focusing cup maintained at the same high
voltage as the filament.
II. Anode
The anode is a target embedded in a copper block that is usually cooled. The
target material is made up of an element that has a high atomic number, high melting
point and a low vapour pressure at the X-ray operating temperature. In an X-ray tube
the anode is kept at a high positive potential in comparison to the filament. When the
filament is heated electrons are generated. These electrons accelerate through the
potential difference between the anode and the cathode. They hit the target and
transfer their kinetic energy to X-ray photons. Only a small amount of the electrons’
kinetic energy produces X-ray photons, while about 99% is converted to heat. This
explains the need for a target material with high melting point. X-ray tube anodes
can be the fixed (stationary) type or the rotating anode type. In this study a fixed
anode X-ray set was used (Figure 8.4 (b)).
III. X-ray tube envelope
The X-ray tube components are engulfed by a tightly air evacuated glass
envelope. When the accelerated electrons generated by the cathode hit the target at
the anode, they transfer their kinetic energy into heat and X-ray photons and the X-
rays leave the X-ray tube case through two or more windows, usually made from
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beryllium as they need to be vacuum tight but highly transparent to X-rays (Figure
8.4).
8.2.3 Microfocus tubes
Microfocus X-ray tubes are used in situations when a fine X-ray beam size is
critical. With a microfocus tube, a high resolution and high magnification is
achievable (Figure 8.4(b)). They are usually demountable X-ray tubes with a very
small focal spot. The focal spot size determines the size of the actual X-ray source.
8.2.4 Electron impact X-ray source
When the accelerated electrons hit the target on the anode they are capable of
producing two different types of radiation; continuous spectrum radiation and
characteristic radiation (Figure 8.5).
FIGURE 8.5 A typical X-ray spectrum produced by a tube with tungsten target
showing continuous and characteristic radiation
I. Continuous radiation
There is a small probability that some electrons from the filament may
penetrate the electron cloud and pass close to or interact with the nucleus or nuclear
field of the target atoms. This interaction involves the deflection of the electron by
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the nucleus accompanied by large energy loss by the electron. This energy is emitted
as high energy electromagnetic X-radiation that is usually referred to as continuous
or Brehmsstrahlung or braking radiation. Continuous radiation contains many energy
levels. When the tube voltage is increased, the intensity of all wavelengths in the
continuous spectrum increases as well as the maximum energy position.
II. Characteristic radiation
This type of radiation occurs simultaneously with Brehmsstrahlung
production. This process involves the interaction of an electron from the filament
with individual orbital electrons in the atoms of the target material. If it has enough
energy, a filament electron may eject an orbital electron from an inner shell (K, L or
M) of the target atom. This is followed by an outer-shell electrons dropping into
inner shells to fill the vacancy, and the difference in energy is emitted in the form of
characteristic radiation. This is called characteristic radiation because it is
"characteristic" for the element and named according to the shell which captured the
electron. For example, characteristic radiation resulting from an outer shell electron
filling a vacant site in the K shell is named K-characteristic radiation.
8.2.5 Factors affecting X-ray beam quantity and quality
X-ray beam quantity usually refers to a measure of the amount or number of
photons in the beam. The words quantity, exposure and intensity are interchangeable
as the higher the quantity or amount of radiation the greater the exposure. On the
other hand, quality of X-ray beam refers to the measurements of its penetrating
power ie. its average photon energy.
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There are many factors affecting the quantity and quality of the final X-ray
beam. These include: tube voltage, tube current, distance from target, target material
and position across the beam and filtration (Figure 8.6).
I. Tube voltage (V)
Increasing the voltage (V) accelerates the electrons emitted from the heated
filament, and the total intensity (I) is proportional to V2:
I α V2 (8.1)
Also the higher the tube voltage, the higher the maximum photon energy will be, and
hence the more penetrative the beam:
Emax α V2 (8.2)
II. Tube current (A)
The total intensity increases on increasing the filament current since this result in
an increase in the tube current which increases the number of electrons hitting the
target:
I A (8.3)
III. Distance from target
There is an inverse square relation between the X-ray intensity (I) and the
distance (d) from the target:
(8.4)
IV. Target material
Target materials with high atomic number (Z) and high density are more efficient
in X-ray production:
I Z (8.5)
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V. Filtration
The purpose of using of filters is to modify the beam spectrum by differential
attenuation of different photon energies. For example in diagnostic radiology the
filters are designed to remove the unwanted low energy photons which will be
otherwise absorbed by the body tissue without contribution to the final radiographic
image.
VI. Summary
Summarising the effect of the above factors on the X-ray spectrum is
illustrated in Figure 8.6, and the X-ray intensity equation can be written as:
(8.6)
FIGURE 8.6 Factors affecting the X-ray spectrum. (a) changing the tube voltage
changes the X-ray spectrum; (b) effect of tube current on the X-ray spectrum; (c)
effect of target material on the spectrum; (d) adding a filter changes the shape of
the X-ray spectrum (Pobe, 1998)
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8.3 X-ray interaction with matter
As an X-ray beam passes through an object, there are three possible ways in
which the photons will react:
I. Penetrate the section of matter without interacting.
II. Interact with the matter and be completely absorbed by depositing their
energy.
III. Interact and be scattered or deflected from their original direction and deposit
part of their energy.
X-rays attenuation depends on the X-rays energy level, density, and atomic number
of the material.
8.3.1 Attenuation mechanisms
The attenuation mechanisms in general of any object are summarised in (Figure
8.7) and described as follows:
I. Photoelectric absorption
The photoelectric interaction involves an interaction between a photon and an
electron from an inner orbital shell at the matter. Usually inner shells electrons bind
firmly to the atom and when their binding energy is only slightly less than the energy
of the photon, they get ejected from the atom and move a relatively short distance
from their original location. The energy transfer is a two-step process; the first step
involves the photoelectric interaction in which the photon transfers its energy to the
electron, the second step involves the electron depositing its energy in the
surrounding matter. The photon's energy is divided into two parts by the interaction.
A portion of the energy is used to overcome the electron's binding energy and to
PART I: INTRODUCTION AND LITERATURE REVIEW
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remove it from the atom. The remaining energy is transferred to the electron as
kinetic energy and is deposited near the interaction site. When the electron is ejected
out of the shell a vacancy is created, usually in shell K or L. This vacancy is then
filled by an electron moving from an outer shell. The difference in energy between
the two shells produces a characteristic X-ray photon (Aichinher et al., 2004, White
and Pharoah, 2004).
II. Compton scattering
Compton scatter occurs when incoming photon has greater energy than the
binding energy of the electron in the atom. As a result only part of the photon energy
is used to eject the electron from its shell (usually outer shell electron). The photon
leaves the site of the interaction in a different direction with reduced energy and the
electron (called recoil electron) distributes its energy via ionisation.
III. Pair production
Pair production is a photon-matter interaction. It takes place when the incident
X-ray has energy greater than 1.02 MeV. The interaction of the incident photon with
the electric field of the nucleus produces an electron-positron pair. This is not a very
common type of interaction and not relevant to this study.
IV. Coherent scattering
In coherent scattering, an incident photon interacts with matter and excites an
atom, causing it to vibrate. The vibration causes the photon to scatter. The coherent
scattering can be also referred to as Thompton scattering.
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I0
x
I
FIGURE 8.7 X-ray attenuation mechanism: (a) Photoelectric effect; (b) Simple scatter;
(c) Compton scatter
8.3.2 X-ray attenuation: Beer’s law
Beer's law, also known as Beer–Lambert law or the Lambert–Beer law
relates the absorption of electromagnetic radiation to the properties of the attenuating
material. A monochromatic X-ray beam is attenuated exponentially as it passes
through a medium (Figure 8.8). This relationship is expressed by Beer’s law as:
I = Io e -µx (8.7)
where
I is the intensity of the attenuated beam
I o is the initial intensity of the beam
x is the thickness of the medium
µ is the linear attenuation coefficient
(LAC)
FIGURE 8.8 Attenuation of a monochromatic X-ray beam of intensity I0 by a homogenous
material thickness x
(a) (b) (c)
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8.3.3 Types of attenuation coefficient (LAC)
I. Linear attenuation coefficient (LAC)
The linear attenuation coefficient (µ) of an element or material refers to the
fraction of the beam of X-rays that is absorbed or scattered per unit thickness of the
material. It has units of cm-1
.
II. Mass attenuation coefficient (MAC)
The mass attenuation coefficient describes the attenuation per unit area density of
material, and has units of m2kg
-1 but is normally expressed as cm
2g
-1. This is because
at a given photon energy, the linear attenuation coefficient can vary significantly for
the same material if it exhibits differences in physical density.
(8.8)
where
µ is the linear attenuation coefficient
p is the density
8.4 X-ray detection
8.4.1 Introduction to semiconductors
There are different types of X-ray detection system, these include: solid state
semiconductor detectors, X-ray films, gas detectors and scintillation detectors. In this
section only the semiconductor detectors are going to be discussed in details, as this
is the type of detector used in this study.
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In semiconductors and insulators the electrons are confined to different bands
of energy and are forbidden from other regions. The band gap represents the energy
difference between the valence band and the conduction band. For semiconductors
the band gap energy is small but not zero with an upper limit of ≈ 4eV, while for
insulators the band gap is large. The main example of these solid state semiconductor
detectors are high purity germanium detectors (HPGe) and lithium drifted silicon
detectors (Si(Li)). A high purity germanium detector is used in this study.
The basic principle behind the operation of semiconductor detectors is that as
the photon passes through the detector, an electron–hole pair is created. These
electron-hole pairs are produced when an electron acquires enough energy to
overcome the band gap and jump from the valence band to the conduction band in
the detector material. Electron–hole pairs are considered the basic information
carriers in solid state detectors (Singh, 2000, Seibert and Boone, 2005).
8.4.2 Multichannel analysers (MCA)
The role of the MCA is to convert the voltage pulses from the detector
preamplifier into digital pulses. These digital pulses are organised in electric “bins”
which correspond to different ranges of voltage pulse. Important characteristics of an
MCA are linearity and stability with respect to temperature changes, and analogue to
digital conversion time. In this study a DSPEC Plus (EG & G ORTEC, TN, USA) is
used as both amplifier and MCA. This MCA system uses a zero dead-time correction
technique developed by ORTEC to correct the actual number of counts by
determining the number of events that must be added to account for pulse pile-up.
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CHAPTER 9
Scanning Microradiography (SMR) Theory and
Methodology
9.1 Introduction
Scanning microradiography is an X-ray attenuation technique which was
initially developed by Elliott et al. (1981). It was later modified by Anderson and
Elliott (1985) to observe real-time physical and chemical changes in specimens. The
aim behind the concept of developing the SMR was to overcome the difficulties
associated with the conventional contact microradiography (CMR) such as
inhomogeneity of the film emulsion due to manufacture variation, saturation of the
photographic emulsion as well as nonlinear response and noise at low X-ray
exposures (Anderson, 1988, Anderson, 1993).
SMR is a point by point X-ray absorption technique which enables
measurement of the intensity of approximately 15 µm transmitted X-ray beams as
they are attenuated by passing through a specimen mounted on the SMR moving
specimen holder stage. The stage has an accuracy of movement of approximately 0.1
µm. It travels a distance of 600 mm horizontally in the X-axis direction and 200 mm
vertically in the Y-axis direction, driven by a stepper motor and controlled by a
computer (Anderson, 1993). The transmitted photons are detected and counted via a
high purity Germanium detector which eliminates the need for close contact between
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specimen and detector. This allows for the creation of separate environmental
chambers enclosing each specimen, which can be altered by, for example, changing
the degree of saturation, chemical composition, pH and the circulating rate of the
solution, all independently of other chambers. SMR allows the study of more than
one specimen simultaneously during an experiment on a single stage. Computer
control of the stage enables the order of scanning of the specimens as well as
parameters of the scanning to be controlled independently. SMR can be considered
the technique of choice for precise measurement of changes in mineral mass as the
experiment conditions can be modified and the effect of the modifications on the
experimented sample can be observed, measured and monitored in real time over a
selected period of time that can be up to 1000 h. The disadvantages of SMR include
that it is much more complex than CMR, it needs a very stable X-ray source and has
a lower spatial resolution, and areas need more time for measurements (Anderson,
1993).
Scanning can be achieved in either a “parallel” or a “perpendicular” direction
depending on the direction of the acid attack in relation to the X-ray beam. When the
acid attack is perpendicular to the central X-ray beam it is called “perpendicular
mode” (Anderson et al., 1998). When the acid attack is parallel to the central beam,
it is called “parallel mode” (Anderson et al., 1998). In this study the “parallel mode”
was used.
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9.2 SMR system apparatus
FIGURE 9.1 SMR machine with its main components X-ray source, X-Y stage,
and detector
FIGURE 9.2 Schematic representation of the SMR system main components and
their connections
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The SMR system apparatus consists of three main components; X-ray generator,
SMR stage with SMR cell’s mounting frame, and X-ray detector (Figure 9.1 and
Figure 9.2).
9.2.1 X-ray generator
SMR requires a very stable X-ray source that demands a high voltage high
stability power supply. An Enraf-Nonious® FR590 X-ray microfocus generator was
used with a PANalytical ® X-ray tube with a silver (Ag) target that gives a
characteristic Kα peak at 22.1 keV (Figure 8.4). An approximately 15 µm aperture
made up from 90% gold and 10% platinum is used to produce an X-ray beam of
approximately 15 µm diameter (Siscoglou, 2008).
9.2.2 X-ray detector
The X-ray detector used is a high purity germanium detector (Ametek, PA,
USA). The detector was coupled to digital spectrometer and multichannel analyser
DSPEC PLUS™ (Digital Gamma-Ray Spectrometer, ORTEC®, Ametec, PA, USA),
which allows spectrum capture (for details of the multichannel analyser refer to
Section 8.4.2). The information in a single voltage (analogue) pulse from a detector
and amplifier is then sent to a digital converter where it is converted into a sequence
of digital values. Counting can be narrowed to only those energy values that fall
within a certain range of energy and therefore monochromatisation of the X-ray
beam can be achieved (Kosoric, 2006).
9.2.3 SMR stage
The SMR apparatus has two stages that move in X and Y directions
(Micromech, UK). The horizontal stage moves in the X-axis direction for a distance
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of 600 mm while the vertical stage moves in the Y-axis direction for a distance of
200 mm. Each stage is controlled by a stepper of 0.1 µm resolution linear encoder
and moved by stepper motors controlled by software (written by Dr P. Anderson,
Queen Mary University of London) and connected to a computer terminal. The
software was designed to enable the stage to perform up to 30 experiments
simultaneously with 30 different parameters (time, number of steps, step size,
standards, etc.).
9.2.4 SMR cells
The SMR cells are made up of polymethyl methacrylate-PMMA. The
dimensions of the cells are 4.0 cm x 5.0 cm. Each cell has a centrally located
chamber of 2.5 cm in diameter and 4.0 mm depth. Each cell has a cover made up of
the same material and dimensions as the cell itself but with 1.0 mm thickness (refer
to Section 10.3 for SMR cell details). Once the SMR cells are ready with the
specimen disc securely positioned in the centre of the chamber, and the covers
securely sealed with silicon and screws, the SMR cells can then be mounted on to the
SMR stage.
9.2.5 Area scanning
Before the main experiment begins, an area scanning of each specimen
should be performed. The area scan gives an indication of the status of the specimen
and its exact location coordinates on the SMR scanning stage (Figure 9.3).
PART I: INTRODUCTION AND LITERATURE REVIEW
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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
S1
S2
S3
S4
S5
S6
S7
S8
S9
S10
S11
S12
S13
S14
S15
S16
0.2-0.4
0-0.2
-0.2-0
-0.4--0.2
Experimental parameters
X start: 242 X step: 1 # X steps: 18 Y start: 68 Y step: 1 # Y steps: 16 Count time: 1 # Scans left: 0 # Scans done: 2 X standard pos: 241 Y standard pos: 78 Stand time: 1 Cell# 1 X or Y: X # points stds 100
FIGURE 9.3 Area scan of an SMR cell with the specimen centrally located where
X and Y axis represents specimen position coordinates on the SMR stage. Two line
scans drawn across the specimen ( ) and scanning parameters are shown on the
side
From the specimen area scan, two horizontal lines are chosen at approximately 2 mm
apart. These lines are called line scans. On each line scan 13 points are chosen. The
points are called scanning positions and refer to the points on the specimen that are
going to be scanned throughout the experiment to determine any change in their
mineral content. The 1st and the last scanning positions are located outside the
specimen and are used as a reference (Io) value.
9.2.6 Data analysis
For the duration of the experiment the 13 scanning positions on each line
scan are continuously scanned and real time counts are detected by the detector. Data
analysis begins by standardising the counts at the chosen point against a standard
measurement which is a point outside the specimen, to correct for variations in X-ray
PART I: INTRODUCTION AND LITERATURE REVIEW
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generator and X-ray counting chain characteristics. According to Beer’s law (Section
8.3.2), for monochromatic radiation the intensity of transmitted beam through a
sample is:
I = Io e -µx
(9.1)
where I is the transmitted X-ray intensity, Io
is the incident X-ray intensity, and x is
the sample thickness and μ is the linear attenuation coefficient.
Knowing the density of the material, ρ, the linear attenuation coefficient is divided
by the density of the material (μ/ρ) and the equation can be written as:
I = Io e -µmM
(9.2)
where µm is the mass absorption coefficient and M is the mass per unit area of the
specimen (g/cm2).
Equation 9.2 can be also written as
[
] (9.3)
where N is the number of transmitted photons and No
is the number of incident
photon, taken outside of the specimen.
Differentiating this gives the error of the m as:
[
√
√ ] (9.5)
√ can be neglected as the number of incident X-ray photons, No, is very high
(≈500,000). N, the number of transmitted X-ray photons, is typically about 50,000
which give a fractional error in m of ≈ 0.5 % (Figure 9.4).
PART I: INTRODUCTION AND LITERATURE REVIEW
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FIGURE 9.4 Example of data analysis and construction of time profile of HAp
mineral mass loss at the scanning positions during the demineralisation process.
The error in each point is of the order of 0.002 g/cm2
Using Equation 9.3, the mass of HAp per unit area (g /cm2) can be calculated by
using the mass attenuation coefficient of HAp (4.69 cm2/g) calculated for AgKα
radiation. At the selected point the X-ray attenuation value can be then converted to
a value for mass of HAp per unit area (g/cm2). Based on the assumption that the
mineral loss in HAp is linear with time, the demineralisation rate can then be
calculated as:
m = at + b (9.4)
where m is the projected mass of HAp per unit area, t is the time, a is the rate of
demineralisation, and b is the intercept on the y axis.
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0 2 4 6 8 10 12 14 16
scanning position
min
eral
mass (
g c
m-2
)
0 h 100 h 200 h 284 h
Scanning position number
PART II: METHODOLOGY
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PART II: METHODOLOGY
PART II: METHODOLOGY
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CHAPTER 10
Modification of Real-time Scanning Microradiography
for the Quantitative Measurements of Dissolution
Kinetics of Compressed Hydroxyapatite Discs over
Short Period of Time
10.1 Introduction
The development of the SMR technique has been on-going for over 20 years,
with several generations. Early versions of SMR had significant drawbacks
particularly associated with the SMR stage. The first drawback was the lengthy
repeat time between measurements of the same point due to the slow movement of
the stage. The second drawback was the low accuracy in stage positioning (5 µm
accuracy).
Later versions of SMR were developed to overcome the problems associated
with the stage by using a commercial X-Y stage. This provided much higher
positioning accuracy, and a faster travel through the X and Y axes, which allowed a
significant increase in sample positioning speed and reduced the length of the repeat
time between successive measurements of the same point. Another improvement in
the later version of SMR was the use of the same computer to control both stage
motion, and detector photon counting system. The later versions were used to study
mineral content changes in specimens over long periods of time and studying
PART II: METHODOLOGY
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multiple scanning positions. The experimental periods of time might extend for
several weeks in order to allow accurate measurements of changes in specimens
mineral mass content.
For the work described in this thesis, modifications to the operation technique
were developed in order to allow the use of SMR for the detection of RDHAp over
short time periods of 24 h or less. In this Chapter, the SMR technique modification
and the development of a new SMR protocol (short scanning protocol) will be
discussed. This short scanning protocol was used in all the experiments described in
this thesis.
10.2 SMR system apparatus used in this study
10.2.1 X-ray generation
An Enraf-Nonius® (now Bruker) FR590 X-ray microfocus generator was
used with a silver (Ag) target PANalytical ®
X-ray tube (Figure 8.4), and was run at 8
mA and 40 kV. The Ag target gives a characteristic Kα peak at 22.1 keV.
A 10 μm aperture (Imaging Equipment, UK) was used with this generator.
The aperture has a cross section of 10 μm ± 0.5 μm, length of 20 μm ± 1 μm and is
constructed from 90% gold and 10% platinum (Figure 10.1). The percentage of X-
rays transmitted decreases the further the distance from the centre of the aperture and
increases with higher energy levels, with 20% transmission at 47 keV, but almost 0%
transmission at 20 keV. The number of SMR measurements is approximately 1800 in
24 h. Therefore a standardisation method was used to correct for variations in the X-
ray generator throughout, and X-ray counting chain characteristics, by scanning a
standard point usually located in the pure polymethyl methacrylate (PMMA) which
PART II: METHODOLOGY
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is unaffected by the experimental setup, by recording data for approximately 30
seconds repeatedly every 10 scan measurements.
FIGURE 10.1 Schematic diagram of the cross section of the aperture assembly D= 10 µm ±
0.5, L= 20 µm ±1.0
10.2.2 X-ray detector
A solid state high purity germanium (HPGe) planar photon detector system
(ORTEC® Ametek, PA, USA) was used for all studies reported in this thesis. This
was an ORTEC® HPGe detector (GLP planar P-type detector) with 0.3 µm ion
implanted window thickness, 0.127 mm beryllium absorbing layers and useful
energy range 3 kV to 300 kV. It was connected to a DSPEC PLUS™ (Digital
Gamma-Ray Spectrometer, ORTEC®, Ametec, PA, USA) which acts as both digital
amplifier and a multi-channel analyser.
The information in a single voltage (analogue) pulse from the detector is
amplified and then sent to an analogue-digital converter where the signal is
converted into a sequence of digital values. Electronic monochromatisation of the
beam was achieved by only counting pulses that fell within a particular energy range.
PART II: METHODOLOGY
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FIGURE 10.2 The main components of the SMR machine including the X-ray source, X-
ray detector, X-Y scanning stage, and the mounting frame with SMR cells
10.2.3 SMR stage
As discussed in Section 9.2.3, the SMR apparatus consists of two
orthogonally mounted stages (Parker Automation, UK) fitted with optical encoders
(Renishaw). The horizontal stage moves in the X-axis direction for a distance of 600
mm, and the vertical stage moves in the Y-axis direction for a distance of 200 mm
(Figure 10.2). The stages are fitted with end of travel and home sensors. They are
controlled and moved by stepper motors with 0.1 μm accuracy linear encoders under
computer control. The computer software was designed to enable the stage to
perform up to 30 experiments simultaneously with 30 different parameters (time,
number of steps, step size, standards, etc.) (Figure 10.2).
PART II: METHODOLOGY
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10.3 Area scanning
The area scanning technique followed in this study is the same as the
standard area scanning technique discussed in Section 9.2.5 and gives the exact
location of the HAp disc coordinates on the SMR scanning stage. However, in this
study, one centrally located point at the centre of the HAp disc was chosen using the
area scanning analysis and used as the scanning position. A second point, the
standard point, was located in the SMR cell wall (PMMA) ie. not in the sample and
therefore unaffected by changes in the experimental setup. The counting time for the
centrally located scanning position was 30 seconds with a standard reading taken
after every 10 measurements. The scanning time for the standard was 30 seconds.
This modification resulted in a much shorter experiment time, as the considerable
movement time between different scanning positions was not required. A large
number of data points (≈ 1800) were obtained resulting in good statistical accuracy
over the 24 h experimental duration.
10.4 Data analysis at a point
Data analysis begins by standardising the count data at the chosen point
against the standard measurement to correct for any variations in the long term X-ray
generation and X-ray counting chain characteristics. According to (Equation 9.2) and
(Equation 9.3) the transmitted photon counts can be converted to mineral mass
content per unit area and accordingly the projected mineral mass content of HAp per
unit area (g/cm2) can be calculated. This is followed by plotting the projected mineral
content as a function of time (Figure 10.3).
PART II: METHODOLOGY
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FIGURE 10.3 Typical example of linear change in projected mineral mass content over the
experimental duration and the calculation of the RDHAp
Based on the assumption that the change in the projected mineral mass
content is linear with time, the demineralisation rate can then be calculated as:
y = a+ bx (10.1)
where y is the projected mass of HAp per unit area, x is the time, b is the rate of
demineralisation, and a is the intercept with the Y-axis.
Accordingly, Figure 10.3 shows that the change in the projected HAp mineral mass
content over 24 h is ≈2% and the RDHAp is 3.17x10-4
g/cm2/h.
10.5 The effect of SMR data sampling frequency on the statistics
of mineral mass loss calculation
One of the main advantages of the SMR technique is that it is designed to
enable the scanning of up to 30 different SMR cells with 30 different scanning
y = -3.17E-04x + 4.12E-01
R2 = 8.45E-01
0.395
0.4
0.405
0.41
0.415
0.42
0 5 10 15 20 25
Time (h)
Min
era
l m
ass (
g/c
m2)
PART II: METHODOLOGY
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parameters simultaneously. The SMR experiments are usually run for several weeks.
This involves a large amount of the data collected over the experimental duration
which allows the scanning of multiple SMR cells simultaneously.
However, in this thesis, a modified SMR technique was developed aiming at
shorter experimental durations (24 h or less), involving a reduction in the number of
data collected to ≈1800 data counts in 24 h.
10.5.1 Effect of even sampling frequency
Figures 10.4, 10.5, 10.6 and 10.7 show the changes in the projected HAp
mineral mass content over 24h at 100%, 50%, 25% and 10% sampling frequencies
respectively.
FIGURE 10.4 Change in the projected HAp mineral mass content over 24 h at
100% sampling frequency
100% Sampling frequencyr2=0.556 StdErr=7.17e-6
b=-0.000332
0 5 10 15 20 25
Time (h)
0.315
0.32
0.325
0.33
0.335
Min
era
l m
ass (
g/c
m2)
PART II: METHODOLOGY
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FIGURE 10.5 Change in the projected HAp mineral mass content over 24 h at 50%
sampling frequency
FIGURE 10.6 Change in the projected HAp mineral mass content over 24 h at 25%
sampling frequency
50% Sampling frequencyr2=0.550 StdErr=1.03e-5
b=-0.000333
0 5 10 15 20 25
Time (h)
0.315
0.32
0.325
0.33
0.335
Min
era
l m
ass (
g/c
m2)
25% Sampling frequencyr2=0.526 StdErr=1.52e-5
b=-0.000329
0 5 10 15 20 25
Time (h)
0.315
0.32
0.325
0.33
0.335
Min
era
l m
ass (
g/c
m2)
PART II: METHODOLOGY
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FIGURE 10.7 Change in the projected HAp mineral mass content over 24 h at 10%
sampling frequency
Table 10.1 summarises the calculated changes in RDHAp, R2 and standard
error (SE), using Microsoft Office Excel 2003® and TableCurve 2D
® programs, at
different sampling frequencies when a HAp disc was exposed to 0.1% acetic acid pH
4.0 demineralisation solution for 24 h.
10% Sampling frequencyr2=0.516 StdErr=2.42e-5
b=-0.000325
0 5 10 15 20 25
Time (h)
0.315
0.32
0.325
0.33
0.335
Min
era
l m
ass (
g/c
m2)
PART II: METHODOLOGY
- 120 -
TABLE 10.1 The RDHAp, R2 and SE calculated at different sampling frequencies using
Microsoft Office Excel 2003® and TableCurve 2D
® programs
Sampling
frequency
RDHAp
(g/cm2/h)
R2
Standard
error
Standard
error in
RDHAp
(%)
calculated
by
Microsoft
Office
Excel
calculated by
TableCurve
2D
calculated
by
Microsoft
Office
Excel
calculated by
TableCurve
2D
data analysis
using
TableCurve
2D
100% 3.32x10-4
3.32x10-4
5.56 x10-1
5.56 x10-1
7.17x10-6
2
50% 3.33x10-4
3.33x10-4
5.49 x10-1
5.50 x10-1
1.03 x10-5
3
33.3% 3.31x10-4
3.31x10-4
5.75 x10-1
5.75 x10-1
1.95 x10-5
6
25% 3.29x10-4
3.29x10-4
5.26 x10-1
5.26 x10-1
1.52 x10-5
5
20% 3.26x10-4
3.26x10-4
5.21 x10-1
5.21 x10-1
1.70 x10-5
5
16.6% 3.31x10-4
3.31x10-4
5.72 x10-1
5.72 x10-1
1.73 x10-5
5
14.3% 3.30x10-4
3.30x10-4
5.48 x10-1
5.50 x10-1
1.93 x10-5
6
12.5% 3.30x10-4
3.30x10-4
5.20 x10-1
5.20 x10-1
2.18 x10-5
7
11.1% 3.25x10-4
3.25x10-4
5.72 x10-1
5.72 x10-1
2.05 x10-5
6
10% 3.25x10-4
3.25x10-4
5.16 x10-1
5.16 x10-1
2.42 x10-5
7
As observed from Table 10.1 the difference in the calculated RDHAp, over
different sampling frequencies ranged between 100% (1800 data counts) and 10%
(180 data counts) using both Microsoft Office Excel 2003® and TableCurve 2D
®,
was 0.07x10-4
g/cm2/h which represents a maximum difference of 2%. Figures 10.4-
10.7 are examples of different sampling frequencies, 100%, 50%, 25% and 10%
respectively. This emphasises the advantage of using the standard error (SE) in
statistical evaluation on the use of R2, since the SE takes in account the sample size
while R2 represent the accuracy of fit.
PART II: METHODOLOGY
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10.5.2 Effect of multiple SMR cells simultaneous scanning
Some of the experiments in this thesis involved the scanning of two or three
SMR cells simultaneously which led to a reduction in the observed data by 50% or
33% respectively. Figures 10.8-10.11 show real-data from simultaneous scanning of
one to four SMR cells respectively. The reduction in the observed data counts was
reflected in a systematic repetitive interrupted pattern (gaps) in the data points.
FIGURE 10.8 Change in the projected HAp mineral mass content over 24 h at 100%
sampling frequency
100% Sampling frequencyr2=0.556 StdErr=7.17e-6
b=-0.000332
0 5 10 15 20 25
Time (h)
0.315
0.32
0.325
0.33
0.335
Min
era
l m
ass (
g/c
m2)
PART II: METHODOLOGY
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FIGURE 10.9 Change in the projected HAp mineral mass content over 24 h at 50%
sampling frequency
FIGURE 10.10 Change in the projected HAp mineral mass content over 24 h at 33%
sampling frequency
50% Sampling frequencyr2=0.567 StdErr=9.56e-6
b=-0.000325
0 5 10 15 20 25
Time (h)
0.315
0.32
0.325
0.33
0.335
Min
era
l m
ass (
g/c
m2)
33% Sampling frequencyr2=0.568 StdErr=1.19e-5
b=-0.000328
0 5 10 15 20 25
Time (h)
0.315
0.32
0.325
0.33
0.335
Min
era
l m
ass (
g/c
m2)
PART II: METHODOLOGY
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FIGURE 10.11 Change in the projected HAp mineral mass content over 24 h at 25%
sampling frequency
As observed in Table 10.2, the difference in the calculated RDHAp over
different sampling frequencies ranged between 3.32x10-4
g/cm2/h when one SMR
cell was scanned at a time and 3.28x10-4
g/cm2/h when three SMR cells were
scanned simultaneously. However, the calculated standard of error shows that 33%
reduction in data counts, scanning three SMR cells simultaneously, lead to
approximately 0.04x10-4
change in SE which supports the reliability of scanning two
or three SMR cells simultaneously.
In this thesis scanning of simultaneous scanning of up to three SMR cells was
used in some of the experiments. TableCurve 2D® was used to demonstrate the
results as it gives a higher level of statistical analysis by calculating the SE for both a
and b (Section 9.2.6) which is more statistically important than R2.
25% Sampling frequencyr2=0.638 StdErr=1.16e-5
b=-0.000337
0 5 10 15 20 25
Time (h)
0.315
0.32
0.325
0.33
0.335
Min
era
l m
ass (
g/c
m2)
PART II: METHODOLOGY
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TABLE 10.2 The RDHAp, R2 and SE calculated at different sampling frequencies
representing multiple SMR cells scanned simultaneously, using Microsoft Office Excel
2003® and TableCurve 2D
® programs
10.6 SMR cell design and specimen preparation
10.6.1 SMR cells
Sampling
frequency
# of
SMR
cells
RDHAp
(g/cm2/h) R2
Standard
error
Standard
error
in RDHAp
(%)
calculated
using
Microsoft
Office
Excel
calculated
using TableCurve
2D
calculated
using
Microsoft
Office
Excel
calculated
using
TableCurve
2D
calculated
using
TableCurve
2D
100% 1 3.32x10-4
3.32x10-4
5.56 x10-1
5.56 x10-1
7.17x10-6
2
50% 2 3.25x10-4
3.25x10-4
5.67 x10-1
5.67 x10-1
9.56 x10-6
3
33.3% 3 3.28x10-4
3.28x10-4
5.68 x10-1
5.68 x10-1
1.19 x10-5
4
25% 4 3.37x10-4
3.37x10-4
6.38 x10-1
6.38 x10-1
1.16 x10-5
3
20% 5 3.19x10-4
3.19x10-4
5.62 x10-1
5.62 x10-1
1.45 x10-5
4
16.6% 6 3.00x10-4
3.00x10-4
5.08 x10-1
5.08 x10-1
1.77 x10-5
6
14.3% 7 3.10x10-4
3.10x10-4
5.48 x10-1
4.71 x10-1
1.71 x10-5
6
12.5% 8 3.50x10-4
3.50x10-4
7.17 x10-1
7.17 x10-1
1.33x10-5
4
11.1% 9 3.00x10-4
3.00x10-4
5.53 x10-1
5.53 x10-1
1.90 x10-5
6
10% 10 3.72x10-4
3.72x10-4
4.02 x10-1
4.02 x10-1
2.51 x10-5
7
FIGURE 10.12 Schematic diagram showing top and side views of the new
design for SMR cells with dimensions
PART II: METHODOLOGY
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FIGURE 10.13 New SMR cell design developed to accommodate fitting of the complete
HAp disc required in this thesis
Modifications of the previous SMR cells designs (either two wells design to
allow experiments using powder or one small chamber design to test sections or
small specimens) was required to enable fitting of the entire HAp disc (Figure 10.12).
An SMR cell with one large (25.0 mm) central chamber was required for this study.
Four SMR cells were prepared from polymethyl methacrylate (PMMA), with
dimensions of 40.0 mm x 50.0 mm. Each cell has a centrally located chamber of 25.0
mm diameter and 4.0 mm depth and a cover made up of the same material as the cell
itself, with the same dimensions but with 1.0 mm thickness (Figure 10.13). Each
SMR cell has two holes on the top and one hole on the bottom. One butterfly needle
is connected to the top hole and one to the hole at the bottom of the SMR cell, to
allow solution to be pumped in and out, maintaining its circulation throughout the
experiment. The butterfly needles are Hospira Venisystems Butterfly® (product #
P293A05, needle length 20.0 mm, needle diameter 0.8 mm). The second top hole
allows the escape of air and prevents building up of internal pressure within the cell
and leakage within the cell in case of pump failure. A single permeable compressed
HAp disc is securely placed in the centre of the SMR cell chamber and the cover is
securely sealed with silicon and screws.
PART II: METHODOLOGY
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10.6.2 Specimen preparation
In this study permeable compressed sintered HAp discs were used as a
representative of dental enamel. The HAp discs were all products of Plasma-Biotal
Ltd, UK, with dimensions of 13.0 mm in diameter x 2.0 mm thickness and 20 wt %
porous (HAp discs type selection will be discussed later in Chapter 11 and Chapter
12). All the compressed HAp discs were preconditioned by the preconditioning
technique followed at the Oral Surface Science Department, School of Oral & Dental
Sciences, University of Bristol, Bristol, UK. The idea behind preconditioning the
HAp discs was to remove any loose particles or more soluble materials on the
surface of the disc. The HAp discs were preconditioned by being submerged in a
beaker containing a stirred solution of citric acid (0.3% normal pH) and turned over
after 15 minutes. The discs were then washed by deionised water and left on filter
paper for few hours to dry. The discs were then coated with acid resistant nail
varnish on all surfaces leaving only one surface exposed so that the acid could
diffuse through into the solid disc. Finally the discs were sterilised by autoclaving
under usual conditions of 121°C (given by 15 p.s.i. pressure from 100% steam) for
30 min sterilisation time.
In this study a single HAp disc was placed in each SMR cell. The HAp disc
was placed in the centre of the SMR cell chamber, covered by the SMR cell cover
which was sealed with silicone rubber compound (RS Components Ltd, Corby,
Northants, UK, product # 692-542) to prevent leakage and tightened up with nine
screws (Figure 10.4). SMR cells were then mounted on the SMR cells mounting
frame on the SMR X-Y stage. The cells were securely mounted on the SMR X-Y
stage by screws, filled and circulated with de-ionised water to keep the specimen
PART II: METHODOLOGY
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hydrated. By this stage, the SMR cells with the HAp discs specimens were ready for
area scanning.
10.7 Demineralisation solutions
10.7.1 0.1% acetic acid pH 4.0
In this study 0.1% acetic acid pH 4.0 was used as representative of caries-like
conditions. In clinical situations dental caries develop in response to organic acids
particularly lactic acid produced by plaque bacteria through fermentation of dietary
carbohydrates. Therefore, in ideal situation lactic acid should have been used to
resemble caries like condition but since lactic acid is quite expensive to obtain and
difficult to find in pure form therefore acetic acid was chosen. Acetic acid has been
used in many studies in this lab and other research centres and it has been shown that
the role of acetic acid in demineralisation is similar to that of lactic acid (Margolis,
1992, Gao et al., 1993, Anderson et al., 2004, Elliott et al., 2005). Acetic acid pH 4.0
was particularly selected because previous SMR studies using acetic acid pH 4.5
required a longer experimental duration to obtain a reliable data as the first 24h data
were noisy.
One litre of 0.1% acetic acid pH 4.0 was prepared from acetic acid 100%
(AnalaR NORMAPUR, VWR International Ltd. England, product # 20104.334,
batch # 08G310506) and de-ionised water (Milipore, Direct-Q5; France). No
additional calcium or phosphate was added and the solution was buffered with 1
Molar HCl or KOH solutions as necessary to reach the targeted pH level. The pH
adjustment was done using Orion-pH/ISE meter Model 710.
PART II: METHODOLOGY
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10.7.2 0.3% citric acid pH 2.8
0.3% citric acid pH 2.8 was used as representative of erosion-like conditions.
This is following protocol used by the Dental Materials Science Laboratory at the
School of Oral and Dental Sciences, University of Bristol in studying dental erosion
in vitro. One litre of 0.3% citric acid pH 2.8 was prepared from citric acid (AnalaR
NORMAPUR, VWR International Ltd. England, product # 100813M, batch #
K91366639 730) and de-ionised water (Milipore, Direct-Q5; France). No additional
calcium or phosphate was added and the solution was buffered with 1 Molar HCl or
KOH solutions as necessary to reach the targeted pH level. The pH adjustment was
done using Orion-pH / ISE meter Model 710.
Each solution was stored separately in one litre bottle, sterilised by
autoclaving at 121°C achieved with 15 psi pressure, 100% steam) and 30 minutes
sterilisation time.
Demineralisation solutions were prepared fresh on the experiment day. When
the same solution was used in a series of experiments on successive days or at
different pH values, the demineralisation solution was made as a bulk solution and
divided into multiple one litre bottles. Each solution was stored separately in one
litre bottle, sterilised by autoclaving at 121°C achieved with 15 psi pressure, 100%
steam) and 30 minutes sterilisation time. The pH was then adjusted on the day of the
experiment. Solutions were circulated at 24 RPM (0.80 ml/min) using Watson
Marlow Pump 205U, UK (Section 14.3). All experiments were carried out at room
temperature, in a thermostatically controlled laboratory (at 22 ± 1°C).
Details of the specific solution used in the experiments are given in the
materials and methods section of each experiment.
- 129 -
PART III: DEVELOPMENT OF A PROTOCOL
PART IV: EXPERIMENTAL WORK
- 130 -
Introduction to Development of a Protocol
The scanning microradiography technique has been previously used to study
the kinetics of enamel and HAp dissolution under erosive and caries simulating
conditions, over a long period of time extending up to 1000 h. However, studying the
kinetics of HAp dissolution over a short period of time (24 h or less) has not been
studied previously using the SMR technique in this laboratory. Therefore, a
development of a protocol was required.
The development of a protocol involved investigating several changeable
parameters regarding the SMR technique, type of HAp discs to be used,
demineralisation solution circulation rate and the concentration of divalent cations.
With regards to the SMR technique, it was modified and tested for its ability
and reliability in detecting RDHAp over a period of 24 h or less (Chapter 13).
In this thesis HAp was used as an analogue for dental enamel (Section 2.5).
Similar studies in this laboratory have mostly used one of two types of HAp discs,
either Plasma-Biotal HAp discs or Hitemco Medical Applications (HIMED) HAp
discs. In order to choose between these two types of discs, they were investigated by
X-ray microtomography (XMT), X-ray diffraction (XRD) and SMR to help in
selecting the most suitable type for this thesis (Chapter 11 and Chapter 12).
The circulation rate of demineralising solution adjacent to a dissolving
surface is an important parameter in SMR experiments since it has a considerable
PART IV: EXPERIMENTAL WORK
- 131 -
influence on the rate of dissolution of solids. Therefore different demineralisation
solution circulation speeds were investigated (Chapter 14).
Finally the effect of Sr2+
in high concentrations was investigated (Chapter
15). Summary of the experiments done to finalise the protocol is given in Table
III.A.
TABLE III.A Experiments performed for developing the thesis protocol
Protocol component Experiment
SMR technique and
duration
Modification of SMR technique to reliably
detect RDHAp over a short period of time
Investigate the demineralisation of compressed
HAp discs with altering acidic buffer with de-
ionised water over short period of time
Selection of HAp discs
Characterisation of HIMED and Plasma-Biotal
compressed HAp discs using X-ray diffraction
and X-ray microtomography
Comparison between HIMED and Plasma-
Biotal compressed HAp discs response (RDHAp)
to exposure to demineralisation solutions using
SMR
Demineralisation solution
circulation speed
Study the effect of demineralisation solution
circulation speed on compressed HAp discs
dissolution kinetics using SMR
Sr2+
concentrations
Study the effect of high concentration
(desensitising toothpaste concentration) of Sr2+
on HAp dissolution kinetics studied using
SMR
PART IV: EXPERIMENTAL WORK
- 132 -
CHAPTER 11
Characterisation of HIMED and Plasma-Biotal
Compressed Hydroxyapatite Discs
11.1 Introduction
In this thesis permeable HAp discs were used as an analogue for dental
enamel. Similar studies in this laboratory have used one of two types of HAp discs.
The first type was Plasma-Biotal Ltd, UK, permeable, compressed and sintered HAp
discs with dimensions of 13.0 mm in diameter x 2.0 mm thickness and 20 wt%
nominal porosity. The second type of HAp disc was the product of Hitemco Medical
Applications, (HIMED), USA, permeable, compressed and sintered HAp discs with
dimensions of 12.05 mm in diameter x 1.25 mm in thickness and 20 wt% nominal
porosity.
11.2 Aims and objectives
The aim of this study was to compare the dissolution behaviour of the
Plasma-Biotal and HIMED permeable compressed HAp discs and select the type of
HAp discs to be used in this thesis.
PART IV: EXPERIMENTAL WORK
- 133 -
The objectives were to investigate the HAp discs purity, uniformity and
porosity using X-ray diffraction (XRD) and X-ray microtomography (XMT)
techniques.
11.3 Materials and methods
11.3.1 X-ray microtomography
Three permeable compressed HAp discs of each type were randomly
selected and scanned using the fourth generation in-house developed XMT system
with a laboratory X-ray generator (Ultrafocus HMX 160, X-Tek system Ltd, 5µm
source, tungsten target, 160 kV) operated at 90 kV and 200µA (Davis and Elliott,
1997).
Two permeable compressed HAp discs were placed flat and fixed by sticky
wax to a Perspex stand that was mounted on the XMT rotation stage and oriented so
that the XMT axis of rotation was as perpendicular as possible to the HAP disc
surface (Figure 11.1)
FIGURE 11.1 HIMED and Plasma-Biotal HAp discs placed flat and fixed on a
Perspex stand with aluminum wire to be mounted on the XMT rotation stage
PART IV: EXPERIMENTAL WORK
- 134 -
11.3.2 X-ray diffraction
Three randomly selected compressed HAp discs of each type were tested for
their mineral content and purity by X-ray diffraction. X-ray diffraction was carried
out using an XPERT-PRO diffractometer system, 1500 W sealed tube with a copper
(Cu) target ran at 40 mA tube current and 45 kV generator voltage to provide CuKα
radiation. The diffraction patterns were then collected from continuous scans ranging
from 5 to 120 2-theta angle.
11.4 Results
11.4.1 XMT
For the XMT, data analysis and visual display of the XMT data set was done
using the Amira™ software package (TGS Template Graphics Software Inc., USA).
The Amira™ program allows visualisation of single slices as well as surface and
volume rendered images enabling viewing of a sample from any angle.
A comparison of the reconstructed images from both types of HAp discs
reveals that they were of evenly uniformity in porosity, whereas HIMED compressed
discs showed greater distribution and larger variety in pores sizes (Figure 11.2 and
Figure 11.3).
PART IV: EXPERIMENTAL WORK
- 135 -
FIGURE 11.2 Reconstructed images of coronal sections through two compressed HAp
discs showing larger pores in upper HAp disc (HIMED (a)) and evenly distributed and sized
pores in lower HAp disc (Plasma- Biotal (b))
FIGURE 11.3 Reconstructed images of axial sections through HIMED HAp discs
(a,b,and c) showing uneven distribution of larger sized pores while Plasma-Biotal
HAp disc (d) shows even distribution of equally sized pores
(a)
A
(d)
A
(c)
A
(b)
A
PART IV: EXPERIMENTAL WORK
- 136 -
11.4.2 XRD
Typical examples of the obtained XRD pattern for the HIMED compressed
HAp discs and the Plasma-Biotal compressed HAp discs are shown in Figure 11.4
and Figure 11.5.
FIGURE 11.4 XRD pattern for HIMED HAp disc from 20-40 (2)
HIMED HAp Disc
0
2000
4000
6000
8000
10000
12000
14000
16000
18000
20 25 30 35 40Diffraction angle 2 theta
En
erg
y level
PART IV: EXPERIMENTAL WORK
- 137 -
Plasma-Biotal HAp Disc
0
2000
4000
6000
8000
10000
12000
14000
16000
18000
20 25 30 35 40
Diffraction angle 2 theta
En
erg
y level
FIGURE 11.5 XRD pattern for Plasma-Biotal HAp disc from 20-40 (2)
FIGURE 11.6 Typical XRD pattern of fully crystalline HAp with principal diffraction peaks
(Prevéy, 2000)
PART IV: EXPERIMENTAL WORK
- 138 -
11.5 Discussion
It was visually apparent from the XMT reconstructed images that the Plasma-
Biotal HAp discs had better uniformity with regard to pore size and distribution,
compared to HIMED HAp discs.
The results shown from the XRD confirmed that both types of HAp discs
contain only hexagonal HAp. When compared to a classical HAp XRD pattern
(Figure 11.6), both HIMED and Plasma-Biotal compressed HAp discs showed a
classical HAp XRD pattern with no additional peaks. However, there is a peak
missing in the Plasma-Biotal XRD results at about 31 degrees 2theta diffraction
angle. There is no explanation to this finding and further research is needed in this
area.
11.6 Conclusion
The narrow and sharp principal diffraction peaks indicate fully crystalline
HAp with no difference in purity and crystal structure between the two types of
discs.
PART IV: EXPERIMENTAL WORK
- 139 -
CHAPTER 12
Comparison of Demineralisation Results of HIMED and
Plasma-Biotal Hydroxyapatite Discs
12.1 Aims and objectives
The aim was to compare the demineralisation rate of HIMED and Plasma-
Biotal HAp discs.
The objective was to measure RDHAp of the two types of HAp discs in
response to exposure to different demineralisation solutions of various pH values
using the SMR technique.
12.2 Materials and methods
12.2.1 SMR
For details of the SMR technique refer to Chapter 10
12.2.2 HAp discs
Two randomly selected HAp discs from each type (HIMED and Plasma-
Biotal) were preconditioned, sterilised and coated with acid resistant nail varnish on
all surfaces except one and positioned in the centre of the SMR cells (for sample
preparation details refer to Section 10.6.2).
PART IV: EXPERIMENTAL WORK
- 140 -
12.2.3 Demineralisation solution
In this study, 4 litres of 0.1% acetic acid solution pH 2.8, 3.2, 3.6 and 4.0
were prepared. Similarly 0.3% citric acid solution was prepared (for demineralisation
solution preparation details refer to Section 10.7). One HAp disc from each type of
discs was exposed to the full series of 0.1% acetic acid demineralisation solutions
(pH 2.8, followed by 3.2, 3.6 and 4.0) for 24 h for each pH value. The HAp disc was
washed with de-ionised water (without pH adjustment) for 30 min between solutions
with different pH values. The same applied to 0.3% citric acid. Each experiment was
duplicated.
12.3 Results
The mineral mass content of each HAp disc was continuously measured
though out the experiment duration. RDHAp was calculated and the results are
summarised in Table 12.1 and Figures 12.1 and 12.2.
TABLE 12.1 RDHAp for both types of HAp discs in response to a change in
demineralisation solution type and pH values
0.1% acetic acid 0.3% citric acid
pH
Plasma-Biotal
disc
RDHAp (g/cm2/h)
HIMED disc
RDHAp (g/cm2/h)
Plasma-Biotal
disc
RDHAp(g/cm2/h)
HIMED disc
RDHAp (g/cm2/h)
2.8
4.44 x 10-4 4.16 x 10-4 1.22 x 10-3 8.58 x 10-4
3.2
4.32 x 10-4 3.67 x 10-4 7.65 x 10-4 4.98 x 10-4
3.6
3.79 x 10-4 3.30 x 10-4 4.72 x 10-4 3.44 x 10-4
4.0
3.69 x 10-4 3.10 x 10-4 4.02 x 10-4 3.39 x 10-4
PART IV: EXPERIMENTAL WORK
- 141 -
FIGURE 12.1 The change in RDHAp for Plasma-Biotal and HIMED HAp discs as a
function of 0.1% acetic acid at a range of pH values
FIGURE 12.2 The change in RDHAp for Plasma-Biotal and HIMED HAp discs as a
function of 0.3% citric acid at a range of pH values
2.5E-04
3.0E-04
3.5E-04
4.0E-04
4.5E-04
5.0E-04
2.6 2.8 3 3.2 3.4 3.6 3.8 4 4.2
pH
Dem
inera
lisati
on
rate
(g
/cm
2/h
)
Plasma-Biotal HIMED
0.0E+00
2.0E-04
4.0E-04
6.0E-04
8.0E-04
1.0E-03
1.2E-03
1.4E-03
2.6 2.8 3 3.2 3.4 3.6 3.8 4 4.2
pH
Dem
inera
lisati
on
rate
(g
/cm
2/h
)
Plasma-Biotal HIMED
PART IV: EXPERIMENTAL WORK
- 142 -
12.4 Discussion
The rate of hydroxyapatite dissolution can be affected by multiple factors
(Section 4.1) among them; the chemical composition of the bulk solid, the pore size
and distribution of the bulk solid, and the pH value of the demineralisation solution.
The use of XMT, XRD and the SMR in the experiments in Chapter 11 and
Chapter 12 was to find the best HAp amongst the two available types to be used in
this thesis.
Based on the results of the XMT study (Chapter 11), the Plasma-Biotal HAp
discs showed better uniformity with regards to pore size and distribution compared
to the HIMED HAp discs. Since larger pores are known to facilitate diffusive
transport of ions, it was expected that the HIMED HAp discs will show faster
demineralisation rates. The results shown in Table 12.1 demonstrate that RDHAp for
the Plasma-Biotal HAp discs was faster than that for the HIMED HAp discs though
they both showed the same pattern in response to change in the pH value of the
demineralisation solution. There is no clear explanation for this observation and
further investigation is required.
12.5 Conclusions
SMR results showed that HIMED HAp discs were less soluble than Plasma-
Biotal HAp discs when exposed to the demineralisation solutions particularly citric
acid. However both discs followed a similar trend in change in RDHAp when
subjected to different demineralisation solutions with different pH values.
Based on the results obtained for the experiments described in Chapter 11
and Chapter 12 it was concluded that both types of discs are made up from fully
PART IV: EXPERIMENTAL WORK
- 143 -
crystalline HAp with no difference in purity or crystal structure and showed a similar
trend in change in RDHAp when subjected demineralisation solutions, however
according the XMT results Plasma-Biotal HAp discs had better uniformity and
porosity than HIMED HAp discs. Therefore it was decided to use Plasma-Biotal
HAp discs in all the experiments in this thesis.
PART IV: EXPERIMENTAL WORK
- 144 -
CHAPTER 13
Demineralisation of Compressed Hydroxyapatite Discs
with Acidic Buffers at a Range of pH Values over Short
Period of Time
13.1 Introduction
In many in vitro studies of model systems for dental caries and erosion, the
solid is usually exposed to demineralising or remineralising solution, but altering
solution conditions involves interrupting the experiment. A major advantage of the
SMR is that the experimental conditions can be altered without interrupting the
experiment. Using the SMR technique in conjunction with pH cycling systems
allows mimicking of pH conditions in the oral cavity (White, 1995, Harless and
Wefel, 2003, Thaveesangpanich et al., 2005).
13.2 Aims and objectives
The main aim of this experiment was to test the ability of the SMR technique
to detect changes in HAp mineral mass content in response to exposure to acidic
buffers at a range of pH values over a short period of time (24 h or less). A further
aim was to investigate whether information could be obtained about the transient
stage between exposure to acid buffer and the de-ionised water.
PART IV: EXPERIMENTAL WORK
- 145 -
The objectives were to obtain reliable quantitative measures of the
demineralisation rate of compressed HAp discs in acidic buffer followed by de-
ionised water using SMR, over periods of 24 h or less.
13.3 Materials and methods
13.3.1 SMR
For details of the SMR technique refer to Chapter 10.
13.3.2 HAp discs
Four HAp compressed discs (Plasma-Biotal, UK) were used in this study. All
discs were preconditioned, sterilised, and painted with acid resistant nail varnish on
all surfaces but one, leaving this surface exposed to the demineralising solution.
Each disc was placed in a separate SMR and mounted in the centre of the SMR cell
chamber. For details of specimen preparation refer to Section 10.6.2.
13.3.3 Demineralisation solutions
In this study 0.1% acetic acid and 0.3% citric acid solutions of pH 2.8, 3.2,
3.6 and 4.0 were buffered with 1M KOH, with no addition of calcium or phosphate.
Each demineralisation solution was stored separately in a 1 litre bottle (for details of
solution preparation refer to Section 10.7). Demineralisation solutions were
circulated through the SMR cell at a slow rate of 0.19cm3/min. The circulation rate
was set at a slow rate to avoid, as much as possible, any mechanical erosion that
might arise from a fast circulation of acidic solutions. The same HAp disc was
exposed to 0.1% acetic acid at pH 2.8 for 20 h followed by 4 h of de-ionised water,
PART IV: EXPERIMENTAL WORK
- 146 -
then 0.1% acetic acid at pH 3.2 for 20 h, followed by 4 h of de-ionised water and
similarly at pH 3.6 and 4.0. The HAp mineral mass content was measured
continuously over the 24 h experiment time. The experiment was repeated with 0.3%
citric acid solution. All experiments were performed in a thermostatically controlled
laboratory at a temperature of 22°C ± 1°C and were duplicated.
13.4 Results
To assess the effect of the acidic buffers at a range of pH values, over a short
periods of time, on RDHAp, the mineral mass content of each HAp disc was
continuously measured over the duration of the experiment of 24 h. Typical
examples of the results obtained are illustrated in Figures 13.1 to 13.8.
13.4.1 0.3% citric acid demineralisation solution
FIGURE 13.1 The change in projected HAp mineral mass content in response to 20 h of
demineralisation by 0.3% citric acid pH 2.8 followed by 4 h of de-ionised water
y = -5.94E-04x + 7.00E-01
R2 = 7.09E-01
y = -1.29E-05x + 6.89E-01
R2 = 3.43E-05
0.682
0.687
0.692
0.697
0.702
0.707
0 5 10 15 20 25Time (h)
Min
era
l m
as
s (
g/c
m2)
citric acid pH 2.8 deionised w ater
PART IV: EXPERIMENTAL WORK
- 147 -
FIGURE 13.2 The change in projected HAp mineral mass content in response to
20 h of demineralisation by 0.3% citric acid pH 3.2 followed by 4 h of de-ionised
water
FIGURE 13.3 The change in projected HAp mineral mass content in response to
20 h of demineralisation by 0.3% citric acid pH 3.6 followed by 4 h of de-ionised
water
y = -4.38E-04x + 6.80E-01
R2 = 5.74E-01
y = -8.94E-06x + 6.72E-01
R2 = 3.70E-05
0.665
0.67
0.675
0.68
0.685
0 5 10 15 20 25
Time (h)
Min
era
l m
ass (
g/c
m2)
citric acid pH 3.2 deionised water
y = -6.84E-06x + 6.60E-01
R2 = 1.09E-05
y = -2.80E-04x + 6.65E-01
R2 = 3.41E-01
0.65
0.655
0.66
0.665
0.67
0 5 10 15 20 25
Time (h)
Min
era
l m
ass (
g/c
m2)
citric acid pH3.6 deionised water
PART IV: EXPERIMENTAL WORK
- 148 -
FIGURE 13.4 The change in projected HAp mineral mass content in response to 20
h of demineralisation by 0.3% citric acid pH 4.0 followed by 4 h of de-ionised
water
y = 7.74E-06x + 6.55E-01
R2 = 1.87E-05
y = -2.37E-04x + 6.60E-01
R2 = 3.20E-01
0.645
0.65
0.655
0.66
0.665
0 5 10 15 20 25Time (h)
Min
era
l m
as
s (
g/c
m2)
citric acid pH 4.0 deionised water
PART IV: EXPERIMENTAL WORK
- 149 -
y = -3.45E-04x + 6.99E-01
R2 = 4.51E-01
y = -3.08E-06x + 6.91E-01
R2 = 3.00E-06
0.685
0.69
0.695
0.7
0.705
0 5 10 15 20 25Time (h)
Min
era
l m
ass (
g/c
m2)
acetic acid pH 2.8 deionised water
13.4.2 0.1% acetic acid demineralisation solution
FIGURE 13.5 The change in projected HAp mineral mass content in response to 20 h of
demineralisation by 0.1% acetic acid pH 2.8 followed by 4 h of de-ionised water
FIGURE 13.6 The change in projected HAp mineral mass content in response to 20 h of
demineralisation by 0.1% acetic acid pH 3.2 followed by 4 h of de-ionised water
y = -2.06E-04x + 6.89E-01
R2 = 2.30E-01
y = -2.11E-06x + 6.85E-01
R2 = 1.39E-06
0.675
0.68
0.685
0.69
0.695
0 5 10 15 20 25
Time (h)
Min
era
l m
ass (
g/c
m2)
acetic acid pH 3.6 deionised water
PART IV: EXPERIMENTAL WORK
- 150 -
FIGURE 13.7 The change in projected HAp mineral mass content in response to 20
h of demineralisation by 0.1% acetic acid pH 3.6 followed by 4 h of de-ionised
water
FIGURE 13.8 The change in projected HAp mineral mass content in response to
20 h of demineralisation by 0.1% acetic acid pH 4.0 followed by 4 h of de-ionised
water
y = -1.67E-04x + 6.78E-01
R2 = 1.64E-01
y = -2.89E-06x + 6.75E-01
R2 = 2.24E-06
0.665
0.67
0.675
0.68
0.685
0 5 10 15 20 25
Time (h)
Min
era
l m
as
s (
g/c
m2)
acetic acid pH 3.6 deionised water
y = -2.63E-05x + 6.75E-01
R2 = 4.01E-01y = -1.80E-06x + 6.75E-01
R2 = 1.58E-04
0.674
0.6745
0.675
0.6755
0.676
0 5 10 15 20 25Time (h)
Min
era
l m
as
s (
g/c
m2)
acetic acid pH 4.0 deionised w ater
PART IV: EXPERIMENTAL WORK
- 151 -
Figure 13.9 summarises the RDHAp for all demineralisation solutions at the
investigated pH range.
FIGURE 13.9 The change in RDHAp in response to changing the demineralisation
solution at a range of pH values
FIGURE 13.10 The change in RDHAp in response to changing the demineralisation
solution at a range of [H+]
y = -2.49E-04x + 1.03E-03
R2 = 9.61E-01
y = -3.15E-04x + 1.45E-03
R2 = 9.61E-01
0.0E+00
1.0E-04
2.0E-04
3.0E-04
4.0E-04
5.0E-04
6.0E-04
7.0E-04
2.4 2.6 2.8 3 3.2 3.4 3.6 3.8 4 4.2
pH
Dem
inera
lisati
on
rate
(g
/cm
2/h
)
0.3% citric acid 0.1% acetic acid
y = 1.84E-01x + 6.66E-05
R2 = 8.72E-01
y = 2.40E-01x + 2.29E-04
R2 = 9.31E-01
0.0E+00
1.0E-04
2.0E-04
3.0E-04
4.0E-04
5.0E-04
6.0E-04
7.0E-04
0 0.0005 0.001 0.0015 0.002
[H+]
Dem
inera
lisati
on
rate
(g
/cm
2/h
)
0.3% citric acid 0.1% acetic acid
PART IV: EXPERIMENTAL WORK
- 152 -
13.5 Discussion
Previously, SMR has been used to provide precise quantitative measurements
of mineral mass changes in real-time in studies measuring the kinetics of
demineralisation and remineralisation of enamel and HAp, particularly over long
periods of time (up to 1000 h). These long periods of experiments (1 week or more)
were required to obtain reliable quantitative kinetic dissolution data. This study has
demonstrated that experimental time can be reduced to 20 h while still obtaining
enough photon counts to obtain reliable data. This was achieved through optimising
the X-ray generator and detection system parameters. In previous experiments, the
generator was usually run at lower tube currents and voltages such as 6 mA and 36
kV or 1.5 mA and 45 kV which according to Equation 8.6 give a relative value of X-
ray intensity (I) of 7776 and 3037 respectively. Increasing the photon energy means
increasing the penetration power of the photons and accordingly increasing the
photons counts. Therefore, in this study, the current and voltage were increased to 8
mA and 39 kV increased the spectrum intensity to 12108 which represent almost
doubling the photon counts (for calculation details refer to Section 8.2.5). By
doubling the photon counts, detection of more data over a shorter period of time was
achievable and accordingly it became possible to obtain more accurate data during
the first 24 h of HAp demineralisation. The results (Figure 13.9) demonstrated that
the linear relationship between the loss of mineral mass content and time (previously
found with longer SMR studies), is also observed by SMR over the shorter duration
used in this study. The essentially linear loss of mineral with time has been attributed
to a surface controlled process of dissolution of the mineral at the advancing front of
the HAp disc.
PART IV: EXPERIMENTAL WORK
- 153 -
A further finding was the instantaneous reduction in the demineralisation rate
of the compressed HAp discs following the change to de-ionised water. This
suggests that the demineralisation process is a surface controlled process rather than
diffusion controlled. If switching from demineralisation solution to de-ionised water
resulted in gradual decrease in RDHAp, and hence a curve seen, this would have
suggested that a diffusion controlled process in which the diffusion of the dissolution
products out the acids and into the compressed HAp disc had an influence on RDHAp.
However, taking in consideration the small size and the porosity of the discs, the
change in the circulating solution will not take more than few minutes to affect the
diffusion whether at the HAp surface or within the pores. Therefore, studying the
transient stage should include a close look at the data of the first few minutes of
change in solutions. This is not possible with the current technique and experiment
methodology. With the amount of data obtained within 1 h or less would be too
noisy and inconclusive. Testing the transient stage is beyond the scope of this
experiment (Bollet-Quivogne et al., 2005, Bollet-Quivogne et al., 2007).
13.6 Conclusions
In conclusion, the study in this chapter has demonstrated that SMR can be
used to quantitatively measure the dissolution of permeable compressed HAp discs
under artificial caries and erosion-like conditions for periods of 24 h or less. This
technique can be used to measure the efficacy of various therapies to reduce the
impact of dental caries and erosion.
PART IV: EXPERIMENTAL WORK
- 154 -
CHAPTER 14
Effect of Circulation Speed of Demineralisation
Solutions on Compressed Hydroxyapatite Discs
Dissolution Rate Studied Using Scanning
Microradiography*
14.1 Introduction
The circulation speed of demineralising solution adjacent to a dissolving
surface has a considerable influence on the rate of dissolution of solids. This is
particularly pertinent to dissolution studies of enamel and similar studies of model
systems for dental caries using compressed hydroxyapatite discs as the substrate.
This chapter summarises the experimental study on the effect of the
circulation speed of demineralisation solution on permeable compressed HAp disc
dissolution kinetics.
14.2 Aims and objectives
The aim of this study was to compare the RDHAp as a function of the
demineralisation solution circulation speed.
* The work described in this chapter was presented at the European Organisation for Caries Research Conference (ORCA), Montpellier, France (September, 2010).
PART IV: EXPERIMENTAL WORK
- 155 -
The objective of this study was to investigate the effect of pumping speed
(solution circulating speed) on compressed HAp discs dissolution rates over a period
of 24 h, using SMR.
14.3 Materials and methods
14.3.1 SMR
For details of the SMR technique refer to Chapter 10.
14.3.2 HAp discs
Three randomly selected compressed HAp discs (Plasma-Biotal, UK) were
used in this study. All discs were preconditioned, sterilised, and painted with acid
resistant nail varnish on all surfaces leaving one surface exposed to the
demineralising solution. Each disc was placed in a separate SMR cell and mounted
in the centre of the SMR cell chamber (Section 10.6.2).
14.3.3 Demineralisation solutions
0.1% acetic acid solution pH 4.0 was used in this study as representative of
dental caries-like conditions. For solution details refer to Section 10.7. The HAp disc
exposed surface was subjected to the demineralising solution for duration of 24 h
followed by 30 min of de-ionised water. The circulation speed was then changed to
the next investigated speed.
14.3.4 Circulating pump
An automatic/manual control multi-channel cassette pump (Watson-Marlow
Bredel pumps, Cornwall UK, model 205U), Figure 14.1 and Figure 14.2, was used
PART IV: EXPERIMENTAL WORK
- 156 -
with orange colour coded tubes (Altec™, Altec Products Limited, Cornwall, UK,
product number116-0532-08, bore size = 0.89 mm) used for pumping solution into
the cell, and blue colour coded tubes (product of Altec™, product number 116-0532-
08, bore size = 1.65 mm) to pump the solution out of the cell. The pump tubes were
then connected to 2.0 m long transmission tube of 1.5 mm diameter, and were
securely connected (via Altec™ barbed straight tubing adapter, product number 05-
44-5513), to butterfly needles (Hospira Venisystems Butterfly®
, product number
P293A05, needle length 20.0 mm, needle diameter 0.8 mm), which were inserted
into the cells as shown in Figure 14.1 and 14.2.
FIGURE14.1 Watson Marlow 205U electric
pump with circulating solution
TABLE 14.1 Manufacturer tubes specifications and flow rate as factor of change
in pumping speed
Tube code Orange / Orange Blue / Blue
Pore size 0.89 mm 1.65 mm
Flow rate at 0.5 RPM 0.016 ml/min 0.043 ml/min
Flow rate at 90 RPM 2.92 ml/min 7.69 ml/min
FIGURE 14.2 The electric pump
connected to the SMR cells via
tubing while the demineralisation
solution circulates into and out of
the SMR cells
PART IV: EXPERIMENTAL WORK
- 157 -
The flow rate at each of the circulating speeds to be used in this experiment
(0, 6, 12, 18, 24, 30, 36 RPM) was measured, using orange-orange tubes, and
calculated in ml/min (Table 14.2).
TABLE 14.2 The measured flow rate in ml/min corresponding to each circulating
speed in RPM.
Parestaltic
pump
speed
(RPM)
0 6 12 18 24 30 36
Measured
flow rate
(ml/min)
0 0.19 0.39 0.58 0.80 0.97 1.17
The demineralising solutions were circulated around the compressed HAp
disc at various circulating speeds of 0, 6, 12, 18, 24, 30, 36 RPM (0, 0.19, 0.39, 0.58,
0.80, 0.97, and 1.17 ml/min respectively). The investigated solution circulation
speeds were chosen in the range of slow speeds in order to keep mechanical erosion
of the surfaces to a minimum and avoid the possibility of cell tube/cell leakage while
maintaining a continuous circulation. Each measurement was repeated in triplicate.
All experiments were run in a thermostatically controlled laboratory at a temperature
of 22°C ± 1°C.
PART IV: EXPERIMENTAL WORK
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14.4 Results
For each of the 21 experiments, the relative mass per unit area of the
compressed HAp disc was measured over the 24 h demineralisation cycle and the
RDHAp was calculated (Table 14.3).
TABLE 14.3 The calculated RDHAP during the exposure to 0.1% acetic acid pH
4.0 at various circulation speeds (in triplicate)
Peristaltic
pump speed
(RPM)
Peristaltic
pump speed
(ml/min)
RDHAp (1)
g/cm2/h
RDHAp (2)
g/cm2/h
RDHAp (3)
g/cm2/h
Mean RDHAp
g/cm2/h
0 0 6.13x10-6
6.85x10-6
6.76x10-6
6.58x10-6
6 0.19 1.20x10-4
1.13x10-4
1.22x10-4
1.18x10-4
12 0.39 1.48x10-4
1.62 x10-4
2.14x10-4
1.70x10-4
18 0.58 2.44x10-4
2.56x10-4
2.42x10-4
2.40x10-4
24 0.80 2.68x10-4
2.92x10-4
2.92x10-4
2.72x10-4
30 0.97 3.07x10-4
3.03x10-4
2.97x10-4
3.13x10-4
36 1.17 3.12x10-4
3.17x10-4
3.19x10-4
3.16x10-4
Typical examples of the real-time change in HAp projected mineral mass
following the exposure to 0.1% acetic acid pH 4.0 with demineralisation solution
circulation speeds between 0.00 and 0.97 ml/min are demonstrated in Figure 14.4
and Figure 14.5 respectively.
PART IV: EXPERIMENTAL WORK
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y=a+bxr2=0.000322 FitStdErr=0.00234
a=0.720
b=-6.13e-06
0 5 10 15 20 25
Time (h)
0.712
0.716
0.72
0.724
0.728M
ine
ral m
ass (
g/c
m2)
FIGURE 14.3 Typical example of the change in projected HAp mineral mass content over
a period of 24 h in response to 0.1% acetic acid pH 4.0 demineralisation solution at 0 ml/min
circulation speed
TABLE 14.4 Statistical analysis, for the data in Figure 14.3, using TableCurve 2D®
Value SE t-value 95% Confidence Limits
a (g/cm2) 0.720 1.067e-04 6748.5202 0.7199 0.7204
b (g/cm2/h) -6.13e-6 7.791e-06 -0.786 -2.141e-5 9.154e-6
( Within 1 SD, 1 SD< < 2 SD, 2 SD< < 3 SD, 3 SD< < 4 SD)
PART IV: EXPERIMENTAL WORK
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y=a+bxr2=0.490 FitStdErr=0.0023
a=0.6843008
b=-0.000307
0 5 10 15 20 25
Time (h)
0.67
0.675
0.68
0.685
0.69M
ine
ral m
ass (
g/c
m2)
FIGURE 14.4 Typical example of the change in projected HAp mineral mass content over
a period of 24 h in response to 0.1% acetic acid pH 4.0 demineralisation solution at 0.97
ml/min circulation speed
TABLE 14.5 Statistical analysis, for the data in Figure 14.4, using TableCurve 2D®
Value SE t-value 95% Confidence Limits
a (g/cm2) 0.684 1.047e-04 6533 0.6840 0.6845
b(g/cm2/h) -3.07e-4 7.71e-06 -42.86 -3.22e-4 - 2.92e-4
( Within 1 SD, 1 SD< < 2 SD, 2 SD< < 3 SD, 3 SD< < 4 SD)
PART IV: EXPERIMENTAL WORK
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The mean RDHAp (g/cm2/h) for the triplicate experiments at each circulation speed
was calculated and plotted against demineralisation solution circulation speed
(ml/min) as demonstrated in Figure 14.5.
FIGURE 14.5 The mean rate of demineralisation (g/cm
2/h) plotted against the
change in demineralisation solution circulation speed (RPM). A curve has been
fitted for viewing purposes only
14.5 Discussion
In this study the demineralising solution circulation speed was altered with
all other factors maintained constant in an attempt to study the effect of circulation
speed on the RDHAp. The selection criteria for the choice of the tested circulation
speed involved; Firstly, the circulation rate should be fast enough to keep the
solution in the SMR cell in state of pseudo constant composition without subjecting
the fine SMR tubes to the danger of leakage/rupture. Secondly, to have a solution
circulation rate that provides minimal possible physical erosion to the HAp disc.
Therefore it was decided to test 0, 6, 12, 18, 24, 30, 36 RPM circulation rates. The
calculated RDHAp of the triplicate experiments at each circulation speed were similar
0.0E+00
5.0E-05
1.0E-04
1.5E-04
2.0E-04
2.5E-04
3.0E-04
3.5E-04
0 6 12 18 24 30 36
Demineralisation solution circulation rate (RPM)
Dem
inera
lisati
on
rate
(g
/cm
2/h
)
PART IV: EXPERIMENTAL WORK
- 162 -
in value with a small standard deviation (Table 14.3). This represents the precision,
repeatability and accuracy of the results.
Figure 14.3 represents a typical example of the effect of 0.1% acetic acid pH
4.0 on the RDHAp when circulated at 0.00 ml/min. When the mineral mass content of
the projected HAp was plotted against time for the 24 h scanning duration, 1800
scanning measurements were recorded. 1708 measurements were within 2 SE
showing a good fit of the data. The data showed a hardly recognisable deceleration
trend in the RDHAp 6.13x10-6
g/cm2/h indicating that when the flow rate was zero, the
compressed HAp discs dissolution rate was minimal. As the compressed HAp disc
dissolves, its dissolution products of calcium, phosphate and hydroxyl ions neutralise
the acidity of the acetic acid and quickly the acid loses its acidic strength.
When the circulation rate was increased to 0.97 ml/min the RDHAp mineral
mass content was measured by 1800 scanning measurements over 24 h. 1718 points
from the obtained data fell within the range of 2 SE.
The mean of the triplicate experiment was 3.13x10-4
g/cm2/h with SE of
5.05x 10-6
. The overall trend showed a linear and consistent regression in HAp
mineral content over 24 h.
Figure 14.5 shows an exponential relationship between the mean RDHAp in
response to changes in the demineralisation solution circulation rate. Comparing the
mean RDHap for each two successive circulation rates reveals that the change in
RDHAp was statistically significant as the demineralisation solution circulation rate
increased from 0 RPM to 6 RPM, from 6 RPM to 12 RPM and from 12 RPM to 18
RPM. The calculated P value for each two successive circulation rates was 0.01,
0.05, and 0.01 respectively. However, as the demineralisation solution circulation
PART IV: EXPERIMENTAL WORK
- 163 -
increased above 18 RPM the change in RDHAp became statistically insignificant with
P values of 1.78, 1.22 and 0.75 for demineralisation solution circulation rate
changing from 18 RPM to 24 RPM, from 24 RPM to 30 RPM and from 30 RPM to
36 RPM respectively.
14.6 Conclusions
This study demonstrates that the solution composition in contact with a
demineralising HAp surface achieved by sufficient circulation speed, or stirring, is
an important parameter in HAp dissolution studies. Diffusive transport of dissolved
substrate away from the dissolving HAp surface will influence the kinetics of the
process.
This study helped in developing the research protocol to be used in the rest of
the experiments in this thesis with regard to selecting the demineralisation solution
circulation speed. It was decided to select 24 RPM (0.80 ml/min) as it was the
highest circulating speed that showed a significant increase in RDHAp.
PART IV: EXPERIMENTAL WORK
- 164 -
CHAPTER 15
Effect of High Concentration of Strontium Ions (Sr2+) on
Hydroxyapatite Dissolution Kinetics Studied Using
Scanning Microradiography
15.1 Introduction
Toothpastes containing Sr2+
were introduced to the market around five
decades ago for the treatment of tooth hypersensitivity. Strontium chloride and
strontium acetate were the most commonly used strontium compounds (Hughes et
al., 2010, Mason et al., 2010). Strontium acetate has the advantage of being
compatible with fluoride (Cummins, 2010). Toothpastes containing 6% and 8%
strontium acetate showed rapid and lasting relief of hypersensitivity (Layer and
Hughes, 2010). The chemical similarity between Sr2+
and Ca2+
made it possible for
Sr2+
to replace Ca2+
, in various structures in the body, including HAp . The effect of
Sr2+
on RDHAp remains an area of controversy (Kikuchi et al., 1994, Bigi et al.,
2007). For further details on Sr2+
background refer to Chapter 6.
15.2 Aims and objectives
The aim of this pilot study was to study the effect of Sr2+
, at concentrations
comparable to those found in desensitising toothpastes, on the dissolution kinetics of
porous HAp discs.
PART IV: EXPERIMENTAL WORK
- 165 -
The objective was to measure the rate of HAp dissolution in permeable HAp
discs using SMR under strictly controlled thermodynamic conditions at Sr2+
concentrations relevant to desensitising toothpastes.
15.3 Materials and methods
15.3.1 HAp discs
Two HAp discs were used in this study. The details of the HAp disc
preparation are described in Section 10.6.2.
15.3.2 Demineralisation solutions
Four solutions were prepared at strontium concentrations reported in
desensitizing toothpastes containing 6% and 8% strontium acetate (Layer and
Hughes, 2010);
1) 1 litre of 0.1% acetic acid pH 4.0 with 6% strontium acetate (SIGMA-
ALDRICH™ product # 388548-500G and batch # 01715JJ).
2) 1 litre of 0.1% acetic acid pH 4.0 with 8% strontium acetate.
3) 1 litre of de-ionised water pH 7.0 with 6% strontium acetate (60,000 ppm)
4) 1 litre of de-ionised water pH 7.0 with 8% strontium acetate (80,000 ppm).
The pH of each solution was adjusted following addition of strontium acetate
by addition of HCl or KOH 1 Molar solutions as necessary. The solutions were
circulated at 0.80 ml/min (Table 14.2).
15.3.3 SMR
SMR Cell 1 contained 1 HAp disc that was exposed to 0.1% acetic acid pH
4.0 with 6% (60,000 ppm) strontium acetate then 0.1% acetic acid pH 4.0 with 8%
PART IV: EXPERIMENTAL WORK
- 166 -
(80,000 ppm) strontium acetate for 40 h each. The two demineralising solution
cycles were separated by 24 hours of de-ionised water.
SMR Cell 2 contained 1 HAp disc exposed to 6% (60,000 ppm) strontium
acetate in de-ionised water followed by 8% (80,000 ppm) strontium acetate for 40 h
each, separated by 24 h of de-ionised water.
15.4 Results
15.4.1 0.1% acetic acid pH 4.0 with 6% strontium acetate
FIGURE 15.1 Increased projected HAp mineral mass content over a period of 40 h
in response to exposure to 0.1% acetic acid pH 4.0 demineralisation solution
containing 6% strontium acetate
The results of the effect of 0.1% acetic acid pH 4.0 with 6% strontium
acetate on RDHAp are shown in Figure 15.1. The RDHAp was stopped and the
projected HAp mineral mass content increased at a rate of 5.14x10-5
g/cm2/h.
y = 5.14E-05x + 8.97E-01
R2 = 6.18E-02
0.887
0.892
0.897
0.902
0.907
0 5 10 15 20 25 30 35 40 45
Time (h)
Min
era
l m
ass (
g/c
m2)
PART IV: EXPERIMENTAL WORK
- 167 -
15.4.2 0.1% acetic acid pH 4.0 with 8% strontium acetate
FIGURE 15.2 Increased projected HAp mineral mass content over a period of 40 h
in response to exposure to 0.1% acetic acid pH 4.0 demineralisation solution
containing 8% strontium acetate
The results of the effect of 0.1% acetic acid pH 4.0 with 8% strontium
acetate on RDHAp are shown in Figure 15.2. The RDHAp was stopped and the
projected HAp mineral mass content increased at a rate of 7.19x10-5
g/cm2/h.
y = 7.19E-05x + 8.33E-01
R2 = 4.58E-02
0.823
0.828
0.833
0.838
0.843
0 5 10 15 20 25 30 35 40 45
Time (h)
Min
era
l m
ass (
g/c
m2)
PART IV: EXPERIMENTAL WORK
- 168 -
15.4.3 De-ionised water pH 7.0 with 6% strontium acetate
FIGURE 15.3 Increased projected HAp mineral mass content over a period of 40 h
in response to exposure to de-ionised water pH7 containing 6% strontium acetate
The results of the effect of de-ionised water pH 7.0 with 6% strontium
acetate on RDHAp are shown in Figure 15.3. The RDHAp was stopped and the
projected HAp mineral mass content increased at a rate of 6.38x10-5
g/cm2/h.
y = 6.38E-05x + 8.58E-01
R2 = 9.01E-02
0.85
0.855
0.86
0.865
0.87
0 5 10 15 20 25 30 35 40 45
Time (h)
Min
era
l m
ass (
g/c
m2)
PART IV: EXPERIMENTAL WORK
- 169 -
15.4.4 De-ionised water pH 7.0 with 8% strontium acetate
FIGURE 15.4 Increased projected HAp mineral mass content over a period of 40 h
in response to exposure to de-ionised water pH7 containing 8% strontium acetate
The results of the effect of de-ionised water pH 7.0 with 8% strontium
acetate on RDHAp are shown in Figure 15.4. The RDHAp was stopped and the
projected HAp mineral mass content increased at a rate of 7.87x10-5
g/cm2/h.
15.5 Discussion
Demineralisation halted when the porous HAp disc was exposed to 0.1%
acetic acid solution pH 4.0 containing either 6% or 8% strontium acetate. Over a
period of 40 hours the mineral mass content of the HAp disc exposed to the
demineralisation solutions actually increased. Similar results were obtained when the
y = 7.87E-05x + 9.15E-01
R2 = 1.17E-01
0.907
0.912
0.917
0.922
0.927
0 5 10 15 20 25 30 35 40 45
Time (h)
Min
era
l m
ass (
g/c
m2)
PART IV: EXPERIMENTAL WORK
- 170 -
HAp disc was exposed to solutions containing 6% and 8% strontium acetate at pH
7.0.
The literature did not reveal any previous demineralisation experiments with
solutions containing high strontium concentrations with which to compare the
results. A possible explanation for the halt in RDHAp and increase in the mineral mass
content suggests that Sr2+
was precipitated on the HAp surface. Another possibility
is that the high Sr2+
concentration in the solution might have affected the X-ray
detection by the detector causing fewer photon counts, reflected as increased mineral
mass content at the HAp disc.
Therefore in order to have good understanding of the effect of Sr2+
on RDHAp
it was decided to test the effect of strontium at low concentrations such as Sr2+
concentrations in water on the HAp dissolution kinetics.
15.6 Protocol summary
Based on the results obtained from Chapters 11-15, a final protocol for the
experiments in this thesis has been developed.
Plasma-Biotal compressed permeable HAp discs will be used as a model for
dental enamel (Chapter 11 and Chapter 12). The HAp discs should be preconditioned
and sterilised (Section 10.6.2) prior to placement at the centre of the SMR cell. The
HAp discs will be scanned using the modified SMR technique for measuring the
RDHAp over a period of 20 h to resemble the oral condition as much as possible while
insuring obtaining enough photon counts for a reliable data. A statistician was
consulted in regards to the sample size. Ideally the larger the sample size the more
statistically sound and reliable the results are, but due to the nature of the SMR
PART IV: EXPERIMENTAL WORK
- 171 -
experiments (length of the experiments and the large number of counts obtained over
20 h) it was justifiable to duplicate the experiments.
Based on the sampling time discussed in Section 10.5, scanning more than
one SMR cell simultaneously would not affect the calculated RDHAp, therefore the
duplicate experiments will be run at the same time by scanning 2 SMR cells
simultaneously. 0.1% acetic acid pH 4.0 demineralisation solution will be used a
representative of caries-like condition and 0.3% citric acid pH 2.8 will be used for
erosion-like conditions. These concentrations have been previously used in published
work by the Dental Physical Sciences Laboratory at Queen Mary, University of
London as well as by the Dental Materials Science Laboratory at the School of Oral
and Dental Sciences, University of Bristol. The demineralisation solutions will be
circulated at 24 RPM (0.80 ml/min) circulation speed (Section 14.6). The three
divalent metal cations to be investigated are Zn2+
, Sr2+
and Cu2+
.
Zinc will be investigated at a range of concentrations relevant to Zn2+
concentrations in dental plaque (0, 5, 10, 15, and 20 ppm) (Section 16.2). Sr2+
will be
investigated at a range of concentrations relevant to Sr2+
concentration in drinking
water (0, 5, 10, 20, and 30 ppm) (Section 17.2) and Cu2+
will be investigated at a
range of concentrations (0, 11.25, 22.50, 45, 90, 150 and 180 ppm) relevant to Cu2+
concentrations that have been investigated in other studies (Section 18.5).
Each cation will be investigated in a series of experiments in an increasing
concentration sequence (e.g. 0, 5, 10, 15, and 20 ppm) or a series of experiments in a
decreasing concentration sequence (e.g. 20, 15, 10, 5, and 0 ppm). All concentrations
in one sequence, increasing or decreasing, should be done on the same HAp disc. For
each cation concentration, the RDHAp will be measured over a period of 20 h
followed by 30 min of washing the HAp disc by de-ionised water at 90 RPM to
PART IV: EXPERIMENTAL WORK
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remove any loosely attached substances, followed by the next concentration for
another 20 h and so on, through the whole series of increasing or decreasing
concentration sequence. The idea behind investigating all different concentrations on
a single HAp disc in a sequence of increasing or decreasing concentration sequence
is an attempt to explore whether the investigated cation exhibits a long lasting effect.
In that case the effect of the different concentrations, represented by RDHAp, in an
increasing concentration sequence will show a different trend (pattern) than the trend
shown by the same concentrations when investigated in a decreasing concentration
sequence. In reverse, if both sequences of cation increasing and decreasing
concentrations showed the same trend of effect on RDHAp regardless of the type of
sequence, this would be an indication that the cation showed a surface effect.
A summary of the final developed protocol for the experiments in this thesis
is shown in Table 15.1.
TABLE 15.1 A summary of the protocol to be used in the SMR studies in this
thesis
Protocol component
Conclusion
SMR technique and
scanning duration
The modified SMR technique for measuring
RDHAp over 24 h or less, to be used in this thesis
Twenty hours of demineralisation is sufficient
to be used as scanning duration and HAp discs
to be washed with de-ionised water at 90 RPM
for 30 min between different experimental
conditions to remove any loosely bound
substances on the surface
Selection of HAp discs Permeable compressed sintered Plasma-Biotal
HAp discs will be used in this thesis as
PART IV: EXPERIMENTAL WORK
- 173 -
representative of dental enamel
Types of demineralisation
solutions
0.1% acetic acid pH 4.0 simulating caries-like
conditions
0.3% citric acid pH 2.8 simulating erosion-like
conditions
Demineralisation solution
circulation rate
24 RPM (0.80 ml/min) demineralisation
solution circulation speed
Zn2+
concentration
Zn2+
will be investigated at concentrations
relevant to Zn2+
concentration in dental plaque
(e.g. 0, 5, 10, 15, and 20 ppm)
Sr2+
concentration
High Sr2+
concentrations such as in
desensitising toothpastes are not suitable for
use in studying RDHAp using the SMR
technique, instead low Sr2+
concentrations
such as Sr2+
concentrations in water (0, 5, 10,
20, and 30 ppm) will be used
Cu2+
concentration
Cu2+
will be investigated at a range of
concentrations (0, 11.25, 22.50, 45, 90, 150
and 180 ppm) relevant to Cu2+
concentrations
that have been investigated in other studies
PART IV: EXPERIMENTAL WORK
- 174 -
PART IV: EXPERIMENTAL WORK
PART IV: EXPERIMENTAL WORK
- 175 -
CHAPTER 16
Effect of Zinc Ions (Zn2+) on Hydroxyapatite
Dissolution Kinetics Studied Using Scanning
Microradiography *
16.1 Introduction
Zinc is a dietary essential trace element that was long ago incorporated in
toothpastes because of its antiplaque activity and ability to reduce calculus formation
as well as oral malodor (background information about Zn2+
was discussed in
Chapter 5). Few studies have been conducted on the direct effects of Zn2+
on HAp
dissolution under either erosion or caries-like conditions. The exact mechanism by
which the Zn2+
divalent metal cation alters HAp dissolution kinetics has been an
issue of controversy (Section 5.6).
16.2 Aims and objectives
The aim of this study was to study the effect of Zn2+
on the dissolution
kinetics of permeable HAp discs, at a range of concentrations relevant to Zn
2+
concentrations in plaque.
* The work described in this Chapter was presented at the 2nd Zinc-UK meeting, London, UK (October 2010) and at the European Organisation for Caries research Conference, Kaunas, Lithuania, (July, 2011)
PART IV: EXPERIMENTAL WORK
- 176 -
The objectives were to measure the RDHAp under strictly controlled
thermodynamic conditions at a range of 0, 5, 10, 15 and 20 ppm Zn2+
over a period
of 20 h using SMR.
16.3 Materials and methods
The general protocol of the experiment is illustrated in Figure 16.1.
16.3.1 HAP discs
Eight HAp discs were used in this study. The details of the HAp disc
preparation were described in Section 10.6.2.
FIGURE 16.1 Schematic diagram of a SMR cell with HAp disc in place connected
to the peristaltic pump (p) for circulating the demineralisation solution over a
period of 20 h followed by 30 minutes of de-ionised water at both increasing ,
and decreasing Zn2+
concentration sequence
PART IV: EXPERIMENTAL WORK
- 177 -
16.3.2 Demineralisation solutions
A 5 litre batch solution of 0.1% acetic acid pH 4.0 was divided into five x 1
litre bottles. Into each, zinc acetate (Fisher Scientific UK Limited, Leicesester, UK,
code # Z/0700/50 and batch # 0951237) was added, so that the final Zn2+
concentration was 0, 5, 10, 15 or 20 ppm. The solution pH was adjusted following
addition of zinc acetate by using 1 Molar HCl or KOH solutions as necessary.
Similarly, a 5 litre batch solution of 0.3% citric acid pH 2.8 was divided into
five 1 litre bottles. Into each, zinc acetate (product of Fisher Scientific UK Limited,
Leicesester, UK, code # Z/0700/50 and batch # 0951237) was added, so that the final
concentration Zn2+
was 0, 5, 10, 15 and 20 ppm. The solution pH was adjusted
following addition of zinc acetate by using 1 Molar HCl or KOH solutions as
necessary (Section 10.7). The demineralisation solutions were circulated at 0.80
ml/min.
16.3.3 SMR
Four HAp discs were fixed centrally in four SMR cells and demineralising
solutions were circulated at 0.80 ml/min. The RDHAp was measured at a single
centrally located point on each disc for approximately 20 h at 22 ± 1°C. Each
experiment was repeated twice for both experiments with increasing, and decreasing
Zn2+
concentration steps. The same HAp disc was used for the entire series of
different Zn2+
concentrations, whether at increasing or decreasing Zn2+
concentration
sequences, with the disc being washed with de-ionised water for 30 min between
each Zn2+
concentrations sequences.
For the increasing Zn2+
concentration sequence 20 h experiments; the HAp
disc was exposed to demineralising solution, with no Zn2+
added; followed by 30
min of washing by de-ionised water, followed by 20 h of exposure to demineralising
PART IV: EXPERIMENTAL WORK
- 178 -
solution with 5 ppm Zn2+
, followed by 30 min of washing by de-ionised water and so
on through all the Zn2+
different concentrations. All steps were performed using the
same HAp disc. In reverse, the decreasing sequence Zn2+
concentration experiments,
the same HAp disc was exposed for 20 h to demineralising solution with, 20 ppm
Zn2+
,
followed by 30 min of washing by de-ionised water, followed by 20 h of
exposure to demineralising solution with 15 ppm Zn2+
, followed by 30 min of
washing by de-ionised water and so on through the decreasing Zn2+
concentrations.
Each experiment was duplicated.
16.4 Results
16.4.1 0.1% acetic acid pH 4.0
For each one of the 20 acetic acid pH 4.0 demineralisation solutions
experiments (containing five different Zn2+
concentrations), the mineral mass loss of
each HAp disc was continuously measured throughout the experimental duration.
Figure 16.2 and Figure 16.3 are typical examples of the real-time change in the
projected HAp mineral mass content in response to exposure to 0.1% acetic acid
solution pH 4.0 with 5 ppm Zn2+
for both increasing and decreasing Zn2+
concentration sequences respectively.
Figure 16.2 shows that the HAp projected mineral mass content decreased
from 0.722 g/cm2 to 0.715 g/cm
2 in 20 h. This reduction represents only a 0.9% loss
of the projected HAp mineral mass over 20 h. While for Figure 16.3 the HAp
projected mineral mass content decreased from 0.691 g/cm2 to 0.684 g/cm
2 in 20 h
which represents only a 1% loss in the HAp projected mineral content over 20 h.
PART IV: EXPERIMENTAL WORK
- 179 -
y=a+bxr2=0.566 FitStdErr=0.0020
a=0.722
b=-0.000389
0 4 8 12 16 20
Time (h)
0.708
0.712
0.716
0.72
0.724
0.728M
ine
ral m
ass (
g/c
m2)
FIGURE 16.2 Typical example of the change in projected HAp mineral mass content over
a period of 20 h in response to 0.1% acetic acid pH 4.0 with 5 ppm Zn2+
demineralisation
solution at increasing Zn2+
concentration sequence
TABLE 16.1 Statistical analysis, for the data in Figure 16.2, using TableCurve 2D®
Value SE t-value 95% Confidence Limits
a (g/cm2) 0.722 1.071e-04 6740.54 0.722 0.722
b (g/cm2/h) -3.89e-4 9.277e-06 -40.34 -3.982e-4 - 3.760e-4
( Within 1 SD, 1 SD< < 2 SD, 2 SD< < 3 SD, 3 SD< < 4 SD)
PART IV: EXPERIMENTAL WORK
- 180 -
y=a+bxr2=0.579 FitStdErr=0.0019
a=0.691
b=-0.000390
0 5 10 15 20
Time (h)
0.68
0.683
0.686
0.689
0.692
0.695M
ine
ral m
ass (
g/c
m2)
FIGURE 16.3 Typical example of the change in projected HAp mineral mass content over a
period of 20 h in response to 0.1% acetic acid pH 4.0 with 5 ppm Zn2+
demineralisation
solution at decreasing Zn2+
concentration sequence
TABLE 16.2 Statistical analysis, for the data in Figure 16.3, using TableCurve 2D®
Value SE t-value 95% Confidence Limits
a (g/cm2) 0.691 1.039e-04 6651.835 0.6908 0.6913
b (g/cm2/h) -3.90e-4 9.001e-06 -43.387 -4.082e-4 - 3.729e-4
( Within 1 SD, 1 SD< < 2 SD, 2 SD< < 3 SD, 3 SD< < 4 SD)
PART IV: EXPERIMENTAL WORK
- 181 -
TableCurve 2D®, automated curve fitting and equation discovery program,
version 5.1 for Windows (SYSTAT®
Software Inc, Richmond CA), was used to
calculate the standard error (SE) for each experiment.The RDHAp was calculated and
the resulting associated errors are summarised in Table 16.3.
TABLE 16.3 RDHAp and calculated SE for each demineralising solution
0.1% acetic acid pH 4.0
RDHAp (g/cm2/h) for increasing Zn2+
concentration sequence
RDHAp (g/cm2/h) for decreasing Zn2+
concentration sequence
HAp
disc1
SE
HAp
disc2
SE
HAp
disc1
SE
HAp
disc2
SE
20
2.97 x10-4
9.30x10-6 2.44 x10-4
8.72x10-6
2.65 x10-4
9.21x10-6 2.50 x10-4
9.21 x10-6
15
3.14 x10
-4 9.11x10-6 2.90 x10
-4 8.90x10-6 2.86 x10
-4 8.78x10-6 2.95 x10
-4 8.78 x10-6
10
3.22 x10
-4 8.81x10-6 3.15 x10
-4 8.69x10-6 3.09 x10
-4 9.56x10-6 3.19 x10
-4 9.54 x10-6
5
3.73 x10
-4 9.25x10-6 3.89 x10
-4 9.00x10-6 3.90 x10
-4 9.27x10-6 3.70 x10
-4 9.30 x10-6
0
4.27x10
-4 8.97x10-6 4.48 x10
-4 8.97x10-6 4.48 x10
-4 9.17x10-6 4.30 x10
-4 9.17 x10-6
Zn
2+ c
on
cen
tra
tio
n (
pp
m)
PART IV: EXPERIMENTAL WORK
- 182 -
16.4.2 0.3% citric acid pH 2.8
Figure 16.4 and Figure 16.5 demonstrate the real-time change of HAp
projected mineral mass following exposure to 0.3% citric acid pH 2.8 solution at a
range of Zn2+
concentrations, for both increasing and decreasing Zn2+
concentration
respectively.
Figure 16.4 shows that the HAp projected mineral mass content decreased
from 0.589 g/cm2 to 0.535 g/cm
2 in 20 h. This reduction represents a 9% loss in
projected HAp mineral mass over 20 h. While for Figure 16.5 the HAp projected
mineral mass content decreased from 0.505 g/cm2 to 0.449 g/cm
2 in 20 h which
represents a 10% loss in the projected mineral content over 20 h.
PART IV: EXPERIMENTAL WORK
- 183 -
FIGURE 16.4 Typical example of the change in projected HAp mineral mass content over a
period of 20 h in response to 0.3% citric acid pH 2.8 with 5 ppm Zn2+
demineralisation
solution at increasing Zn2+
concentration sequence
TABLE 16.4 Statistical analysis, for the data in Figure 16.4, using TableCurve 2D®
Value SE t-value 95% Confidence Limits
a (g/cm2) 0.589 1.87e-04 3143.845 0.5882 0.5889
b (g/cm2/h) -2.77e-3 1.62e-05 -170.807 -2.80e-3 - 2.74e-3
y=a+bxr2=0.953 FitStdErr=0.00357
a=0.589
b=-0.00277
0 4 8 12 16 20
Time (h)
0.52
0.53
0.54
0.55
0.56
0.57
0.58
0.59
0.6M
ine
ral m
ass (
g/c
m2)
( Within 1 SD, 1 SD< < 2 SD, 2 SD< < 3 SD, 3 SD< < 4 SD)
PART IV: EXPERIMENTAL WORK
- 184 -
FIGURE 16.5 Typical example of the change in projected HAp mineral mass content over a
period of 20 h in response to 0.3% citric acid pH 2.8 with 5 ppm Zn2+
demineralisation
solution at decreasing Zn2+
concentration sequence
TABLE 16.5 Statistical analysis, for the data in Figure 16.5, using TableCurve 2D®
Value SE t-value 95% Confidence Limits
a (g/cm2) 0.505 2.53e-04 1994.060 0.5048 0.5057
b (g/cm2/h) -2.85e-3 2.20e-05 -129.974 -2.90e-3 - 2.81e-3
y=a+bxr2=0.915 FitStdErr=0.0050
a=0.505
b=-0.00285
0 4 8 12 16 20
Time (h)
0.44
0.45
0.46
0.47
0.48
0.49
0.5
0.51
0.52M
ine
ral m
ass (
g/c
m2)
( Within 1 SD, 1 SD< < 2 SD, 2 SD< < 3 SD, 3 SD< < 4 SD)
PART IV: EXPERIMENTAL WORK
- 185 -
The change in RDHAp after the sequential exposure to 0.3% citric acid pH 2.8
with various Zn2+
concentrations was calculated and the results obtained are
summarised in Table 16.6.
TABLE 16.6 RDHAp and calculated SE for each demineralising solution
0.3% citric acid pH 2.8
RDHAp (g/cm2/h) for increasing Zn
2+
concentration sequence
RDHAp (g/cm2/h) for decreasing Zn
2+
concentration sequence
HAp
disc1
SE
HAp
disc2
SE
HAp
disc1
SE
HAp
disc2
SE
20
1.70x10-3
1.65x10-5 1.96x10-3
1.48x10-5
1.59x10-3
1.46x10-5 1.74x10-3
1.40 x10-5
15 2.51x10-3
1.87x10-5 2.38x10-3
1.54x10-5 2.45x10-3
1.76x10-5 2.42x10-3
1.73 x10-5
10 2.84x10-3
2.19x10-5 2.62x10-3
1.60x10-5 2.60x10-3
1.58x10-5 2.68x10-3
1.74 x10-5
5 2.89x10-3
2.36x10-5 2.77x10-3
1.62x10-5 2.85x10-3
2.20x10-5 2.88x10-3
2.25 x10-5
0 3.18x10-3
2.15x10-5 3.06x10-3
1.73x10-5 2.90x10-3
2.26x10-5 2.95x10-3
2.18 x10-5
Zn
2+ c
on
cen
tra
tio
n (
pp
m)
PART IV: EXPERIMENTAL WORK
- 186 -
16.5 Discussion
Previous studies on the effect of Zn2+
on de/remineralisation of enamel
concluded that Zn2+
interacts with the HAp either through adsorbing onto the surface
of the crystals or through incorporation into the crystal lattice replacing Ca2+
and
forming zinc calcium phosphates (Xu et al., 1994, Stötzel et al., 2009)
In this study, for caries-like conditions, Figure 16.2 and Figure 16.3 represent
typical examples of the change in projected HAp mineral mass content, over a period
of ≈20 h when exposed to 0.1% acetic acid pH 4.0 with 5 ppm Zn2+
demineralisation
solution with increasing and decreasing concentration sequences respectively. In
Figure 16.2 the change in mineral mass content (g/cm2) was plotted as a function of
time (h). The data showed a linear regression trend for the projected HAp mineral
mass content over time. One thousand and five hundred data counts were measured
at a centrally located point on the permeable HAp disc over 20 h, of which only 76
data counts were outside 2 SD (5%).
Figure 16.3 shows demineralisation in caries-like conditions similar to those
in Figure 16.2 but in the sequence when the Zn2+
concentration experiments had
been reversed. It shows a similar linear regression trend in projected HAp mineral
mass content over the experimental duration. One thousand five hundred data counts
were collected at a centrally located point on the permeable HAp disc over 20 h out
of which 50 data counts were outside 2 SD (3.3%).
Table 16.3 shows the calculated demineralisation rates and the SE for each of
the 20 experiments with various Zn2+
concentrations. Calculations of SE gives a
better insight into the accuracy of the data than R2, particularly when dealing with
large data sets as it takes into consideration the sample size while R2 only
represents
PART IV: EXPERIMENTAL WORK
- 187 -
a measure of goodness of fit. The calculated SE for the fitted parameters were low,
as demonstrated in Table 16.3.
Figure 16.6 shows that as Zn2+
concentration increased at an increasing
concentration sequence (0-20 ppm), the RDHAp decreased. This reduction in RDHAp
was statistically significant (P≤0.05) for all Zn2+
concentrations investigated when
compared to the control group (0 ppm). However, when the sequence of Zn2+
concentrations was reversed (20-0 ppm), the RDHAp increased (Figure 16.7).
PART IV: EXPERIMENTAL WORK
- 188 -
FIGURE 16.6 The effect of Zn2+
at a range of 0 – 20 ppm on mean RDHAp at increasing Zn2+
concentration sequence under caries-like conditions
FIGURE 16.7 The effect of Zn2+
at a range of 20 - 0 ppm on mean RDHAp at decreasing Zn2+
concentration sequence under caries-like conditions
2.0E-04
2.5E-04
3.0E-04
3.5E-04
4.0E-04
4.5E-04
5.0E-04
0 5 10 15 20 25
Zinc concentration (ppm)
Dem
inera
lisati
on
rate
(g
/cm
2/h
)
2.0E-04
2.5E-04
3.0E-04
3.5E-04
4.0E-04
4.5E-04
5.0E-04
0 5 10 15 20 25
Zinc concentration (ppm)
Dem
inera
lisati
on
rate
(g
/cm
2/h
)
PART IV: EXPERIMENTAL WORK
- 189 -
0.0E+00
1.0E-04
2.0E-04
3.0E-04
4.0E-04
5.0E-04
0 5 10 15 20
Zinc concentration (ppm)
De
min
era
lis
ati
on
ra
te (
g/c
m2/h
)
mean increasing sequense mean decreasing sequence
4.38x10-4
2.60x10-4
3.76x10-4
3.19x10-4 3.02x10-4
2.71x10-4
3.19x10-4 2.99x10-4
4.34x10-4
3.81x10-4
The average of each duplicate experiment, at each Zn2+
concentration, in both
increasing and decreasing Zn2+
concentration sequence was calculated and illustrated
in Figure 16.8.
FIGURE 16.8 The effect of 0.1% acetic acid pH 4.0 with different Zn2+
concentrations
(ppm) on RDHAp (g/cm2/h) at both increasing and decreasing concentration sequences
Figure 16.8 shows that the relation between Zn2+
concentration and RDHAp is
the same for both, increasing and decreasing concentration sequences. An important
outcome of this study is that the direction of the sequence of Zn2+
concentration has
no effect on its capability to reduce RDHAp. This is as if Zn2+
was completely washed
away when the HAp disc was rinsed by the de-ionised water between the different
concentrations in each sequence. This supports the hypothesis that Zn2+
is not
permanently incorporated into the HAp structure; but instead adheres to the HAp
surface blocking dissolution nuclei and slowing the demineralisation rate.
PART IV: EXPERIMENTAL WORK
- 190 -
For erosion-like conditions, Figure 16.4 and Figure 16.5 are typical examples
of the change in projected HAp mineral mass content over a period of 20 h during
exposure to 0.3% citric acid pH 2.8 with 5 ppm Zn2+
demineralisation solution
during an increasing and a decreasing concentration sequences respectively.
Figure 16.4 shows a regression trend for the projected HAp mineral mass
content over time. One thousand five hundred data counts were measured at a
centrally located point on the permeable HAp disc over 20 h, of which only 55 data
counts were outside 2 SD (3.6%).
Figure 16.5 shows demineralisation in erosion-like conditions similar to
those for Figure 16.4 but with the sequence of the Zn2+
concentration experiments
reversed. It shows a similar regression trend in projected HAp mineral mass content
over the experimental duration. One thousand five hundred data counts were
collected at a centrally located point on the permeable HAp disc over 20 h, out of
which only 62 data counts were outside 2 SD (4.1%).
Table 16.4 shows the calculated demineralisation rates and the SE for each of
the 20 experiments in which various Zn2+
concentrations were used.
Figure 16.9 shows the effect of Zn2+
on RDHAp at an increasing concentration
sequence (0-20 ppm), that RDHAp decreased. This reduction in RDHAp was
statistically significant (P≤0.05) for all Zn2+
concentrations investigated when
compared to the control group (0 ppm). However, when the sequence of Zn2+
concentrations was reversed, as Zn2+
concentrations decreased (20-0 ppm), the
RDHAp increased (Figure 16.10).
PART IV: EXPERIMENTAL WORK
- 191 -
FIGURE 16.9 The effect of Zn
2+ at a range of 0 – 20 ppm on mean RDHAp at increasing Zn
2+
concentration sequence under erosion-like conditions
FIGURE 16.10 The effect of Zn2+
at a range of 20 – 0 ppm on mean RDHAp at decreasing
Zn2+
concentration sequence under erosion-like conditions
0.0E+00
5.0E-04
1.0E-03
1.5E-03
2.0E-03
2.5E-03
3.0E-03
3.5E-03
0 5 10 15 20 25
Zinc concentration (ppm)
Dem
inera
lisati
on
rate
(g
/cm
2/h
)
0.0E+00
5.0E-04
1.0E-03
1.5E-03
2.0E-03
2.5E-03
3.0E-03
3.5E-03
0 5 10 15 20 25Zinc concentration (ppm)
dem
inera
lisati
on
rate
(g
/cm
2/h
)
PART IV: EXPERIMENTAL WORK
- 192 -
0.0E+00
5.0E-04
1.0E-03
1.5E-03
2.0E-03
2.5E-03
3.0E-03
3.5E-03
0 5 10 15 20
Zinc concentration (ppm)
Dem
inera
lisati
on
rate
(g
/cm
2/h
)
mean increasing sequence mean decreasing sequence
3.12x10-3
2.95x10-3 2.83x10-3 2.88x10-3
2.73x10-3 2.68x10-3
2.45x10-3 2.42x10-3
1.74x10-3 1.83x10-3
FIGURE 16.11 The effect of 0.3% citric acid pH 2.8 with different Zn2+
concentration (ppm) on RDHAp (g/cm2/h) at both increasing and decreasing
concentrations sequences
Figure 16.11 shows the relation between Zn2+
concentration and RDHAp is the
same for both increasing and decreasing concentration sequences. An important
outcome of this study is that it demonstrated that the sequence of Zn2+
concentration
in a series of experiments has no effect on its ability in reducing RDHAp, i.e. Zn2+
were completely washed away when the HAp disc was rinsed by de-ionised water
between the different concentrations. This supports the hypothesis that Zn2+
does not
incorporate into the HAp structure; instead it adheres to the surface blocking some
dissolution nuclei and slowing the demineralisation rate.
An overall comparison between the results of the effect of Zn2+
on RDHAp in
caries and erosion-like conditions clearly indicates that both showed a decrease in
RDHAp with increasing Zn2+
concentrations. All solutions with a range of Zn2+
concentrations (5, 10, 15 and 20 ppm) showed a significant decrease (p ≤ 0.05) in
RDHAp compared to the control solution (0 ppm Zn2+
). This suggests that Zn2+
is in a
PART IV: EXPERIMENTAL WORK
- 193 -
“loose equilibrium” with the HAp surface mineral, and therefore while there is Zn2+
in the surrounding fluid some will be adsorbed onto the surface in a dynamic
equilibrium. This finding is in agreement with Tan-Walker and Gilbert (1989), who
showed that Zn2+
reduced demineralisation significantly at physiologically relevant
zinc concentrations added to a gel acid demineralisation system.
16.6 Conclusions
The results of this study demonstrated the inhibitory effect of Zn2+
as a
divalent metal cation on RDHAp under strictly controlled thermodynamic conditions
relevant to dental caries and erosion. The results also support the hypothesis that
Zn2+
(under the experimental conditions) inhibits HAp dissolution by adsorbing to
the surface of the HAp disc rather than having a substitution effect.
PART IV: EXPERIMENTAL WORK
- 194 -
CHAPTER 17
Effect of Strontium Ions (Sr2+) at a Range of
Concentrations (0-30 ppm) on Hydroxyapatite
Dissolution Kinetics Studied Using Scanning
Microradiography*
17.1 Introduction
Numerous clinical trials have reported the efficacy of a wide range of Sr2+
containing compounds in the management of dentine hypersensitivity. The British
and American Dental Associations have accredited various formulations for efficacy,
including toothpastes incorporating strontium acetate and strontium chloride
(Orchardson and Gillam, 2006). On the other hand the role of Sr2+
in the prevention
of dental caries shows many controversies. Experimental studies show that the
replacement of Ca2+
by Sr2+
alter the HAp crystal lattice, and the formed strontium
calcium apatite is more soluble than the HAp. However clinical studies showed that
populations who lived in areas with high Sr2+
water concentration level had higher
Sr2+
concentration in their enamel and experienced less dental caries than those from
areas with lower Sr2+
water concentration level (Curzon and Crocker, 1978, Curzon
et al., 1978, Athanassouli et al., 1983, Curzon, 1985).
* The work described in this chapter was presented at the International Association of Paediatric Dentistry Conference, Athens, Greece, (June, 2011) and at the British Society of Oral and Dental Research, Sheffield, UK (September 2011).
PART IV: EXPERIMENTAL WORK
- 195 -
17.2 Aims and objectives
The aim of this study was to investigate the effect of Sr2+
at concentrations of
0, 5, 10, 20 and 30 ppm on the dissolution kinetics of permeable HAp disc.
The objectives were to measure the rate of HAp dissolution of a permeable
HAp disc using the SMR technique under strictly controlled thermodynamic
conditions and Sr2+
range of concentrations relevant to concentrations found in
drinking water supplies.
17.3 Materials and methods
The protocol of this experiment is illustrated in Figure 17.1.
FIGURE 17.1 Schematic diagram of an SMR cell with HAp disc in place,
connected to the peristaltic pump (p) for circulating the demineralisation so lution
over a period of 20 h followed by 30 minutes of de-ionised water at both increasing
, and decreasing Sr2+
concentration sequences
1 2
5 4 3 2 1
5 4 3
PART IV: EXPERIMENTAL WORK
- 196 -
17.3.1 HAp discs
Eight HAp discs were used in this study. The details of the HAp disc
preparation were described in Section 10.6.2.
17.3.2 Demineralising solutions
For cariogenic conditions, a 5 litre batch solution of 0.1% acetic acid pH 4.0
was divided into five 1 litre bottles. Into each one, strontium acetate (SIGMA-
ALDRICH, Co., St. Louis, USA, product # 388548-500G and batch # 01715JJ
SIGMA-ALDRICH™) was added, so that the final Sr2+
concentration was 0, 5, 10, 20
and 30 ppm Sr2+
.
For erosive conditions, a 5 litre batch solution of 0.3% citric acid pH 2.8
was divided into five 1 litre bottles. Into each one, strontium acetate was added, so
that the final Sr2+
concentration was 0, 5, 10, 20 and 30 ppm Sr2+
.
After the addition of strontium acetate, the pH of each demineralising
solution was adjusted by using 1 Molar HCl or KOH solutions as necessary (Section
10.7).
17.3.3 SMR
HAp discs were located centrally in the SMR cells and demineralising
solutions were circulated at 0.80 ml/min (Chapter 14). The rate of HAp
demineralisation was measured at a centrally located point in each disc for a ≈20 h at
22 ± 1°C. Each experiment was repeated twice in both increasing (0 - 30 ppm) and
decreasing (30 - 0 ppm) Sr2+
concentrations sequence.
For the increasing Sr2+
concentration experiments; the HAp disc was exposed
for ≈20 h to the demineralising solution with no Sr2+
added; followed by 30 min of
washing by de-ionised water, followed by ≈20 h of exposure to demineralising
solution with 5 ppm Sr2+
, followed by 30 min of washing by de-ionised water and so
PART IV: EXPERIMENTAL WORK
- 197 -
on through the increasing Sr2+
concentrations. All exposures were performed using
the same HAp disc. In reverse, for the decreasing sequence Sr2+
concentration
experiments, the same HAp disc was further exposed for ≈ 20 h to each
demineralising solution with 30 min of washing by de-ionised water. The SMR cells
were mounted on the SMR stage and scanned at the same time. Each experiment was
duplicated.
17.4 Results
17.4.1 0.1% acetic acid pH 4.0
For each experiment of the 20 demineralisation experiments using 0.1%
acetic acid pH 4.0 with various Sr2+
concentrations, the mineral mass loss of each
HAp disc was continuously measured throughout the entire experimental duration.
Figure 17.2 and Figure 17.3 are typical examples of the real-time change in the
projected HAp mineral mass content in response to the exposure to 0.1% acetic acid
pH 4.0 solution with 20 ppm Sr2+
concentration in both increasing and decreasing
Sr2+
concentration sequences respectively.
For Figure 17.2 the HAp projected mineral mass content decreased from
0.776 g/cm2 to 0.772g/cm
2 in 20 h. This reduction in projected mineral mass content
represents only a 0.5% loss of projected HAp mineral content over 20 h. While for
Figure 17.3 the HAp projected mineral mass content decreased from 0.677 g/cm2 to
0.675 g/cm2
in ≈20 h which represents 0.3% loss of projected mineral content over
≈20 h at a rate of 1.05x10-4
g/cm2/h.
PART IV: EXPERIMENTAL WORK
- 198 -
FIGURE 17.2 Typical example of the change in projected HAp mineral mass content over
a period of ≈ 20 h in response to 0.1% acetic acid pH 4.0 with 20 ppm Sr2+
demineralisation
solution at increasing Sr2+
concentration sequence.
TABLE 17.1 Statistical analysis, for the data in Figure 17.2, using TableCurve 2D®
Value SE t-value 95% Confidence Limits
a (g/cm2) 0.776 1.651e-04 4697.25 0.7756 0.7762
b (g/cm2/h) -1.82e-4 1.35e-05 -13.55 -2.09-4 - 1.57e-4
y=a+bxr2=0.234 FitStdErr=0.00216
a=0.776
b=-0.000182
0 5 10 15 20 25
Time (h)
0.767
0.77
0.773
0.776
0.779
0.782M
ine
ral m
ass (
g/c
m2)
( Within 1 SD, 1 SD< < 2 SD, 2 SD< < 3 SD, 3 SD< < 4 SD)
PART IV: EXPERIMENTAL WORK
- 199 -
FIGURE 17.3 Typical example of the change in projected HAp mineral mass content over a
period of 20 h in response to 0.1% acetic acid pH 4.0 with 20 ppm Sr2+
demineralisation
solution at decreasing Sr2+
concentration sequence
TABLE 17.2 Statistical analysis, for the data in Figure 17.3, using TableCurve 2D®
Value SE t-value 95% Confidence Limits
a (g/cm2) 0.677 1.747e-04 3878.92 0.6773 0.6780
b (g/cm2/h) -1.05e-4 1.331e-05 -7.924 -1.312e-4 - 7.935e-4
y=a+bxr2=0.067 FitStdErr=0.00251
a=0.677
b=-0.000105
0 5 10 15 20 25
Time (h)
0.665
0.67
0.675
0.68
0.685M
ine
ral m
ass (
g/c
m2)
( Within 1 SD, 1 SD< < 2 SD, 2 SD< < 3 SD, 3 SD< < 4 SD)
PART IV: EXPERIMENTAL WORK
- 200 -
The RDHAp and the SE for each of the 20 experiments, using 0.1% acetic acid
pH 4.0, was calculated and the results associated errors were summarized in Table
17.3
TABLE 17.3 RDHAp and SE for each demineralisation solution at different Sr2+
concentrations at both increasing and decreasing concentration sequences
0.1% acetic acid pH 4.0
RDHAp (g/cm2/h) increasing Sr
2+
concentration sequence
RDHAp (g/cm2/h) decreasing Sr
2+
concentration sequence
HAp
disc1
SE
HAp
disc2
SE
HAp
disc1
SE
HAp
disc2
SE
30
1.30 x10-4 1.28x10-5 1.00 x10-4 1.18x10-5
1.76 x10-4 1.31x10-5 1.18 x10-4 1.28 x10-5
20 1.82 x10-4 1.51x10-5 1.05 x10-4 1.43x10-5 1.42 x10-4 1.69x10-5 1.06 x10-4 1.43 x10-5
10 2.05 x10-4 1.23x10-5 1.71 x10-4 1.57x10-5 1.18 x10-4 1.65x10-5 9.03 x10-5 1.30 x10-5
5 2.80 x10-4 9.61x10-5 2.66 x10-4 1.25x10-5 9.00 x10-5 1.28x10-5 3.19 x10-5 1.75 x10-5
0 3.20x10-4 1.25x10-5 3.60 x10-4 1.38x10-5 2.14 x10-4 1.09x10-5 2.63 x10-4 1.35 x10-5
Sr2
+ c
on
cen
trati
on
(p
pm
)
PART IV: EXPERIMENTAL WORK
- 201 -
17.4.2 0.3% citric acid pH 2.8
Figure 17.4 and Figure 17.5 are typical examples of the real-time change in
the HAp projected mineral mass content in response to the exposure to 0.3% citric
acid pH 2.8 solution with 20 ppm Sr2+
concentrations in both increasing and
decreasing Sr2+
concentrations respectively.
FIGURE 17.4 Typical example of the change in projected HAp mineral mass content over a
period of 20 h in response to 0.3% citric acid pH 2.8 with 20 ppm Sr2+
demineralisation
solution at increasing Sr2+
concentration sequence
TABLE 17.4 Statistical analysis, for the data in Figure 17.4, using TableCurve 2D®
Value SE t-value 95% Confidence Limits
a (g/cm2) 0.432 2.026e-04 2132.276 0.4317 0.4325
b (g/cm2/h) -3.66e-3 1.613e-05 -226.720 -3.689e-3 - 3.626e-3
y=a+bxr2=0.988 FitStdErr=0.00271
a=0.432
b=-0.00366
0 5 10 15 20 25
Time (h)
0.35
0.36
0.37
0.38
0.39
0.4
0.41
0.42
0.43
0.44
Min
era
l m
ass (
g/c
m2)
( Within 1 SD, 1 SD< < 2 SD, 2 SD< < 3 SD, 3 SD< < 4 SD)
PART IV: EXPERIMENTAL WORK
- 202 -
FIGURE 17.5 Typical example of the change in projected HAp mineral mass content over a
period of 20 h in response to 0.3% citric acid pH 2.8 with 20 ppm Sr2+
demineralisation
solution at decreasing Sr2+
concentration sequence
TABLE 17.5 Statistical analysis, for the data in Figure 17.5, using TableCurve 2D®
Value SE t-value 95% Confidence Limits
a (g/cm2) 0.578 1.779e-04 3248.48 0.5774 0.5781
b (g/cm2/h) -3.38e-3 1.508e-05 -216.711 -3.298e-3 - 3.239e-3
y=a+bxr2=0.987 FitStdErr=0.00237
a=0.578
b=-0.00338
0 5 10 15 20 25
Time (h)
0.5
0.51
0.52
0.53
0.54
0.55
0.56
0.57
0.58
0.59M
ine
ral m
ass (
g/c
m2)
( Within 1 SD, 1 SD< < 2 SD, 2 SD< < 3 SD, 3 SD< < 4 SD)
PART IV: EXPERIMENTAL WORK
- 203 -
For each of the 20 demineralisation experiments using 0.3% citric acid pH
2.8 with various Sr2+
concentrations, the projected mineral mass loss of each HAp
disc was continuously measured throughout the entire experiment duration. The
RDHAp and the SE were calculated and the results obtained are summarized in Table
17.6
TABLE 17.6 The RDHAp and SE for each demineralisation solution at different
Sr2+
concentrations in both increasing and decreasing concentration sequences
0.3% citric acid pH 2.8
RDHAp (g/cm2/h) increasing Sr
2+
concentration sequence
RDHAp (g/cm2/h) decreasing Sr
2+
concentration sequence
HAp
disc1
SE
HAp
disc2
SE
HAp
disc1
SE
HAp
disc2
SE
30
3.37 x10-3 1.43x10-5 2.87 x10-3 1.38x10-5
3.57 x10-3 1.41x10-5 4.30 x10-3 1.56 x10-5
20 3.66 x10-3 1.61x10-5 3.24 x10-3 1.46x10-5 3.38 x10-3 1.51x10-5 3.72 x10-4 1.23 x10-5
10 3.75 x10-3 1.95x10-5 3.41 x10-3 1.27x10-5 3.21 x10-3 1.65x10-5 3.17 x10-3 1.34 x10-5
5 4.08 x10-3 1.36x10-5 3.95 x10-3 1.55x10-5 2.60 x10-3 1.19x10-5 2.56 x10-3 1.65 x10-5
0 4.31x10-3 1.46x10-5 4.12 x10-3 1.22x10-5 5.42 x10-3 1.32x10-5 5.88 x10-3 1.65 x10-5
Sr2
+ c
on
cen
trati
on
(p
pm
)
PART IV: EXPERIMENTAL WORK
- 204 -
17.5 Discussion
In this study, Sr
2+ at drinking water supply concentration levels was
investigated at a range between 5 ppm and 30 ppm, based on the literature,
particularly the work done by Little and Barrett (1976), Curzon et al. (1978),
Athanassouli et al. (1983), Featherstone et al. (1983a), Curzon (1985) and Thuy et
al. (2008).
The results of strontium 0 ppm solution were used as a control for
comparison of the effect of different Sr2+
concentrations. Following the same
experiment protocol used in the Zn2+
experiments, a series of demineralisation
solutions was used, that differed only in the Sr2+
concentration, in either an
increasing or a decreasing concentration sequence on the same permeable HAp disc.
Running the experiments in a series of five experiments (≈20 h each), separated by
30 min of washing by stirred de-ionised water removed any loosely adsorbed
material from the surface to evaluate the persistence effect of Sr2+
.
Figure 17.2 and Figure 17.3 represents typical examples of the change in
projected HAp mineral mass content, over a period of ≈20 h when exposed to 0.1%
acetic acid pH 4.0 for caries-like conditions with 20 ppm Sr2+
demineralisation
solution in increasing and decreasing concentration sequences respectively. The data
showed a linear regression trend for the projected HAp mineral mass content over
time. The systematic gaps in the recording of data over the experimental duration are
because more than one SMR cell was scanned simultaneously over the experimental
duration. As discussed in Chapter 10, the SMR technique utilizes a large number of
data points to obtain good statistical accuracy. Scanning more than one SMR cell
requires considerable move time, so bunches of data points were collected for each
cell, but this does not affect the calculated RDHAp (Chapter 10).
PART IV: EXPERIMENTAL WORK
- 205 -
In Figure 17.2, 606 data counts were measured at a centrally located point on
the permeable HAp disc over ≈20 h, of which only 28 data counts were outside 2 SD
(4.6%).
Figure 17.3 shows demineralisation in caries-like conditions similar to those
in Figure 17.2 but with the sequence of the Sr2+
concentration experiments reversed.
It shows a similar linear regression trend for the projected HAp mineral mass content
over the experimental duration. Six hundred and six data counts were collected at a
centrally located point on the permeable HAp disc over ≈20 h out of which 24 data
counts were outside 2 SD (3.9%).
Table 17.3 shows the calculated demineralisation rate and the SE for each of
the 20 experiments with various Sr2+
concentrations.
The effect of Sr2+
on RDHAp at increasing concentrations of Sr2+
sequence
showed that as Sr2+
concentration increased in the range from 0-30 ppm, RDHAp
decreased (Figure 17.6). The reduction in RDHAp was statistically significant
(P≤0.05) for all Sr2+
concentrations investigated when compared to the control group
(0 ppm). While the effect of Sr2+
on RDHAp at decreasing concentrations of Sr2+
sequence showed that as Sr2+
concentration decreased in the range from 30 - 0 ppm,
the RDHAp continued to decrease significantly, except for 0 ppm where the mean
RDHAp increased (Figure 17.7).
PART IV: EXPERIMENTAL WORK
- 206 -
FIGURE 17.6 The effect of Sr2+
at a range of 0 -30 ppm on mean RDHAp at increasing Sr2+
concentration sequence under caries-like conditions
FIGURE 17.7 The effect of Sr2+
at a range of 30 - 0 ppm on mean RDHAp at decreasing Sr2+
concentration sequence under caries-like conditions
0.0E+00
1.0E-04
2.0E-04
3.0E-04
4.0E-04
0 5 10 15 20 25 30 35
Strontium concentration (ppm)
Dem
inera
lisati
on
rate
(g
/cm
2/h
)
0.0E+00
1.0E-04
2.0E-04
3.0E-04
4.0E-04
0 5 10 15 20 25 30 35
Strontium concentration (ppm)
Dem
inera
lisati
on
rate
(g
/cm
2/h
)
PART IV: EXPERIMENTAL WORK
- 207 -
The average of each duplicate experiment, at each Sr2+
concentration at
increasing and decreasing Sr2 concentration sequence was calculated and shown in
Figure 17.8.
FIGURE 17.8 The effect of 0.1% acetic acid pH 4.0 with different Sr2+
concentrations
(ppm) on RDHAp (g/cm2/h) at both increasing and decreasing concentrations sequences
Figure 17.8 shows that Sr2+
had an inhibitory effect on the RDHAp. The
reduction in RDHAp was statistically significant with P≤0.05 for all Sr2+
concentrations when compared to the control group (0 ppm Sr2+
concentration). It
also shows that the reduction in RDHAp was affected by the sequence of Sr2+
concentration. When the permeable HAp disc was exposed to caries simulating
conditions containing 10 ppm Sr2+
at increasing Sr2+
concentration sequence the
mean RDHAp was 1.88x10-4
g/cm2/h. However when the same experiment was
repeated in a decreasing concentration sequence, the mean RDHAp was 1.04x10-4
g/cm2/h. A similar observation was seen for all investigated Sr
2+ concentrations. It
was observed that among the investigated Sr2+
concentrations, the maximum
0.0E+00
1.0E-04
2.0E-04
3.0E-04
4.0E-04
0 5 10 20 30
Strontium concentration (ppm)
De
min
era
lis
ati
on
ra
te (
g/c
m2/h
)
mean increasing sequence mean decreasing sequence
3.40 x 10-4
2.73 x 10-4
1.88 x 10-4
1.44 x 10-4
1.15 x 10-4
2.39 x 10-4
6.10 x 10-5
1.04 x 10-4
1.24 x 10-4
1.47 x 10-4
PART IV: EXPERIMENTAL WORK
- 208 -
reduction in RDHAp in increasing Sr2+
concentrations sequence experiments was
achieved using 30 ppm Sr2+
while for the decreasing Sr2+
concentration sequence
experiments the maximum reduction in RDHAp was achieved using 5 ppm Sr2+
. This
supports the idea that Sr2+
replaces Ca2+
in the HAp crystal lattice and forming a
different crystal phase (strontium-calcium-phosphate) which has a more permanent
effect.
For erosion-like conditions Figure 17.4 and Figure 17.5 represents typical
examples of the change in projected HAp mineral mass content over a period of ≈20
h when exposed to 0.3% citric acid pH 2.8 demineralisation solution containing 20
ppm Sr2+
at increasing and decreasing concentration sequence respectively. The data
showed a linear regression trend for the projected HAp mineral mass content over
time. The systematic periodic interruption in recording the data over the
experimental duration is because of more than one SMR cell been scanned
simultaneously over the experimental duration. Figure 17.4 shows that 606 data
counts were counted at a centrally located point on the permeable HAp disc over ≈20
h out of which only 25 data counts were outside 2 SD (4.1%). The HAp projected
mineral mass content decreased at approximately 10 times faster rate than in caries-
like conditions. It decreased from 0.432 g/cm2 to 0.355 g/cm
2 in ≈20 h. This
reduction in projected HAp mineral mass content represent a 17.8% loss in projected
HAp mineral content over ≈20 h. This further supports that caries is a slowly
progressing disease while erosion involves a rapid loss of dental enamel.
Figure 17.5 represents the demineralisation in erosion-like conditions similar
to those in Figure 17.4 but with the Sr2+
concentration sequence reversed. It shows a
similar linear regression trend in projected HAp mineral mass content over the
experimental duration. Six hundred and six data counts were counted at a centrally
PART IV: EXPERIMENTAL WORK
- 209 -
located point on the permeable HAp disc over ≈20 h out of which only 30 data
counts were outside 2 SD (4.9%). The HAp projected mineral mass content
decreased from 0.578 g/cm2 to 0.513g/cm
2 in ≈20 h which represents 11.4% loss of
projected mineral content over ≈20 h.
Table 17.4 shows the calculated demineralisation rate and the SE for each of
the 20 experiments with various Sr2+
concentrations under erosion-like conditions.
The mean effect of Sr2+
on RDHAp at increasing concentration sequence
showed that as Sr2+
concentration increased in the range from 0-30 ppm, the RDHAp
decreased (Figure 17.9). The reduction in RDHAp was statistically significant P≤0.05
for all Sr2+
concentrations investigated when compared to the control group (0 ppm).
While the mean effect of Sr2+
on RDHAp at a decreasing concentration sequence
showed that as Sr2+
concentration decreased in the range from 30 - 5 ppm, the RDHAp
continued to decrease significantly except for 0 ppm where the mean RDHAp
increased (Figure 17.10).
PART IV: EXPERIMENTAL WORK
- 210 -
2.5E-03
3.0E-03
3.5E-03
4.0E-03
4.5E-03
0 5 10 15 20 25 30 35
Strontium concentration (ppm)
Dem
iner
alis
atio
n r
ate
(g/c
m2/h
)
mean increasing sequence
FIGURE 17.9 The effect of Sr2+
at a range of 0 - 30 ppm on mean RDHAp at increasing Sr2+
concentration sequence under erosion-like conditions
FIGURE 17.10 The effect of Sr2+
at a range of 30 - 0 ppm on mean RDHAp at decreasing
Sr2+
concentration sequence under erosion-like conditions
2.5E-03
3.0E-03
3.5E-03
4.0E-03
4.5E-03
0 5 10 15 20 25 30 35
Strontium concentration (ppm)
Dem
inera
lisati
on
rate
(g
/cm
2/h
)
mean decreasing sequence
PART IV: EXPERIMENTAL WORK
- 211 -
0.0E+00
1.0E-03
2.0E-03
3.0E-03
4.0E-03
5.0E-03
0 5 10 20 30Strontium concentration (ppm)
De
min
era
lis
ati
on
ra
te (
g/c
m2/h
)
mean increasing sequence mean decreasing sequence
The average of each duplicate experiments, at each Sr2+
concentration, at
both increasing and decreasing Sr2+
concentration sequence was calculated and
shown in Figure 17.11.
Figure 17.11 The effect of 0.3% citric acid pH 2.8 with different Sr2+
concentrations (ppm)
on RDHAp (g/cm2/h) at both increasing and decreasing concentrations sequences
Similar to caries-like conditions, Figure 17.11 shows that Sr2+
had an
inhibitory effect on the RDHAp. The reduction in RDHAp was statistically significant
with (P ≤ 0.05) for all Sr2+
concentrations when compared to the control group (0
ppm Sr2+
concentration) and the reduction in RDHAp was affected by the sequence of
Sr2+
concentration in the experimental series. It was observed that among the
investigated Sr2+
concentrations, the maximum reduction in RDHAp in increasing Sr2+
concentrations sequence experiments was achieved using 30 ppm Sr2+
while for the
decreasing Sr2+
concentration sequence experiments the maximum reduction in
4.22 x 10-3
3.65 x 10-3 4.02 x 10-3
2.58 x 10-3
3.58 x 10-3
3.19 x 10-3
3.45 x 10-3 3.55 x 10-3
2.12 x 10-3
3.94 x 10-3
PART IV: EXPERIMENTAL WORK
- 212 -
RDHAp was achieved using 5 ppm Sr2+
, in support of the hypothesis that Sr2+
replaces
Ca2+
in the HAp crystal lattice forming different crystal phase with longer lasting
effect on the apatite dissolution. This can be clinically interpreted as better to give a
larger dose of a Sr2+
containing therapeutic agent (30 ppm of Sr2+
) initially, and then
provide lower maintenance doses of 5 ppm.
Comparison of the results of the effect of Sr2+
on RDHAp under caries and
erosion-like conditions shows that they both shared similar regression trend in
RDHAp in response to an increase in Sr2+
concentrations. The results of this study also
confirm that dental caries involves slowly progressive loss of mineral content while
erosion involves a faster loss of mineral content (≈10 times faster), which can be
explained by the nature of the effect of the two different acids as well as the
difference in pH.
17.6 Conclusions
In conclusion, the addition of Sr2+
decreased RDHAp under strictly controlled
thermodynamic conditions relevant to both dental caries and erosion. However, this
decrease was not reversed when the Sr2+
concentration was subsequently decreased.
This pattern of influence of Sr2+
suggests a partial inclusion of Sr2+
into the HAp
lattice.
PART IV: EXPERIMENTAL WORK
- 213 -
CHAPTER 18
Effect of Copper Ions (Cu2+) on Hydroxyapatite
Dissolution Kinetics Studied Using Scanning
Microradiography
18.1 Introduction
Copper is an essential element required for many normal body functions
such as red blood cell synthesis, collagen cross linking as well as metabolism and
production of energy.
Copper has been reported to be associated with low caries prevalence in
animals such as rats, as well as in human beings. Its caries inhibitory property has
been attributed mainly to its antimicrobial effect against oral bacteria associated with
dental caries (Section 7.2).
The direct effect of copper ions on hydroxyapatite dissolution has not been
studied as extensively as its antimicrobial effect (Section 7.4). There is still much
uncertainty about the exact mechanism through which copper increases dental
enamel resistance against acid attacks. For further details about copper please refer
to Chapter 7.
PART IV: EXPERIMENTAL WORK
- 214 -
18.2 Aims and objectives
The aim of this study was to investigate the effect of Cu2+
at a range of
concentrations of 0 to 180 ppm on the dissolution kinetics of permeable HAp disc.
The objective was to measure the rate of HAp dissolution of a permeable
HAp disc using the SMR under strictly controlled thermodynamic conditions
relevant to dental caries and erosion at a range of Cu2+
concentrations relevant to
those used in other studies.
18.3 Materials and methods
The protocol of this experiment is illustrated in Figure 18.1.
FIGURE 18.1 Schematic diagram of an SMR cell with HAp disc in place,
connected to the peristaltic pump (p) for circulating the demineralisation solution
over a period of 20 h followed by 30 minutes of de-ionised water at both increasing
, and decreasing Cu2+
concentration sequences
PART IV: EXPERIMENTAL WORK
- 215 -
18.3.1 HAp discs
Eight HAp discs were used in this study. The details of the HAp discs
preparation were described in Section 10.6.2.
18.3.2 Demineralising solutions
For cariogenic conditions, a 7 litre batch solution of 0.1% acetic acid pH 4.0
was divided into seven 1 liter bottles. Into each one, copper sulphate (SIGMA-
ALDRICH™, Product code # 1000950043 and batch # 070M0268V) was added, so
that the final Cu2+
concentrations were 0, 11.25, 22.50, 45, 90, 150, and 180 ppm.
For erosive conditions, a 7 litre batch solution of 0.3% citric acid pH 2.8 was
divided into seven 1 liter bottles. Into each one, copper sulphate was added, so that
the final Cu2+
concentration were of 0, 11.25, 22.50, 45, 90, 150, and 180 ppm.
After the addition of copper sulphate, the pH of each solution was adjusted by
using 1 Molar HCl or KOH solutions as necessary (Section 10.6).
18.3.3 SMR
HAp discs were located centrally in the SMR cells and demineralising
solutions were circulated at 0.80 ml/min. The rate of HAp demineralisation was
measured at a centrally located point in each disc for ≈20 h at 22 ± 1°C. Each
experiment was repeated for both increasing (0-180 ppm) and decreasing (180-0
ppm) Cu2+
concentration sequences.
For the increasing Cu2+
concentration experiments, the HAp disc was exposed
for ≈20 h to demineralising solution with no Cu2+
added, followed by 30 min of
washing by de-ionised water, followed by ≈20 h of exposure to demineralising
solution with 11.25 ppm Cu2+
, followed by 30 min of washing by de-ionised water
and so on through the increasing Cu2+
concentrations. All exposures were performed
using the same HAp disc (Figure 18.1). In reverse, for the decreasing Cu2+
PART IV: EXPERIMENTAL WORK
- 216 -
concentration experiments HAp disc was exposed for ≈20 h to each demineralising
solution with 30 min of washing by de-ionised water. The SMR cells were mounted
on the SMR stage and scanned simultaneously. Each experiment was duplicated.
18.4 Results
18.4.1 0.1% acetic acid pH 4.0
For each experiment of the 28 demineralisation experiments using 0.1% acetic
acid pH 4.0 with various Cu2+
concentrations, the mineral mass loss of each HAp
disc was continuously measured throughout the entire experimental duration. Figure
18.2 and Figure 18.3 are typical examples of the real-time change in projected HAp
mineral mass content in response to exposure to 0.1% acetic acid pH 4.0 solution
with 22.5 ppm Cu2+
concentration in both increasing and decreasing Cu2+
concentration respectively.
Figure 18.2 shows that the projected HAp mineral mass content decreased
from 0.671 g/cm2 to 0.667 g/cm
2 in 20 h. This reduction represents only a 0.5% loss
in the projected HAp mineral mass over 20 h at a rate of 1.41x10-4
g/cm2/h. Such
subtle changes are difficult to detect and measure without a powerful technique of
high precision such as the SMR technique. While for Figure 18.3 the HAp projected
mineral mass content decreased from 0.642 g/cm2 to 0.639 g/cm
2 in ≈20 h which
represents a 0.5 % loss in the projected mineral mass over ≈20 h at a rate of 2.1x10-4
g/cm2/h.
PART IV: EXPERIMENTAL WORK
- 217 -
y=a+bxr2=0.124 FitStdErr=0.00234
a=0.670
b=-0.000141
0 5 10 15 20 25
Time (h)
0.66
0.665
0.67
0.675
0.68M
ine
ral m
ass (
g/c
m2)
FIGURE 18.2 Typical example of the change in projected HAp mineral mass content over
a period of ≈20 h in response to 0.1% acetic acid pH 4.0 with 22.5 ppm Cu2+
demineralisation solution at increasing Cu2+
concentration sequence
TABLE 18.1 Statistical analysis, for the data in Figure 18.2, using TableCurve 2D®
Value SE t-value 95% Confidence Limits
a (g/cm2) 0.670 1.760e-04 3809.40 0.6701 0.6708
b (g/cm2/h) -1.41e-4 1.52e-05 -9.26 -1.70-4 - 1.11e-4
( Within 1 SD, 1 SD< < 2 SD, 2 SD< < 3 SD, 3 SD< < 4 SD)
PART IV: EXPERIMENTAL WORK
- 218 -
y=a+bxr2=0.208 FitStdErr=0.00243
a=0.642
b=-0.000210
0 5 10 15 20 25
Time (h)
0.63
0.635
0.64
0.645
0.65M
ine
ral m
ass (
g/c
m2)
FIGURE 18.3 Typical example of the change in projected HAp mineral mass content over
a period of ≈20 h in response to 0.1% acetic acid pH 4.0 with 22.5 ppm Cu2+
demineralisation solution at decreasing Cu2+
concentration sequence
TABLE 18.2 Statistical analysis, for the data in Figure 18.2, using TableCurve 2D®
Value SE t-value 95% Confidence Limits
a (g/cm2) 0.642 1.830e-04 3509.40 0.6111 0.6418
b (g/cm2/h) -2.10e-4 1.57e-05 -12.59 -2.42-4 - 1.89e-4
( Within 1 SD, 1 SD< < 2 SD, 2 SD< < 3 SD, 3 SD< < 4 SD)
PART IV: EXPERIMENTAL WORK
- 219 -
The RDHAp and the SE for each of the 28 experiments, using to 0.1% acetic
acid pH 4.0 were calculated and the results obtained were summarized in Table 18.3.
TABLE 18.3 RDHAp and SE for each demineralisation solution at different Cu2+
concentrations at both increasing and decreasing concentration sequences
0.1% acetic acid pH 4.0
RDHAp (g/cm2/h) increasing Cu
2+
concentration sequence
RDHAp (g/cm2/h) decreasing Cu
2+
concentration sequence
HAp
disc1
SE
HAp
disc2
SE
HAp
disc1
SE
HAp
disc2
SE
180
9.40x10-5 2.88x10-5 7.45 x10-5 1.57x10-5
8.55 x10-5 2.23x10-5 8.01 x10-5 2.53 x10-5
150
9.50 x10-5 1.30x10-5 1.10 x10-4 1.10x10-5 1.56 x10-4 1.19x10-5 1.31 x10-4 1.13 x10-5
90
1.14 x10-4 1.68x10-5 1.23 x10-4 1.23x10-5 1.73 x10-4 1.43x10-5 1.51 x10-4 1.53 x10-5
45
1.26 x10-4 1.48x10-5 1.40 x10-4 1.40x10-5 1.93 x10-4 2.15x10-5 1.74 x10-4 1.88 x10-5
22.5
1.41 x10-4 1.52x10-5 1.88 x10-4 1.88x10-5 2.98 x10-4 1.85x10-5 2.10 x10-4 1.57x10-5
11.25 2.30 x10-4 1.55x10-5 2.61 x10-4 2.61x10-5 2.33 x10-4 1.26x10-5 2.61 x10-4
1.30 x10-5
0 3.68 x10-4 1.49x10-5 3.36 x10-4 3.36x10-5 3.20 x10-4 1.65x10-5 3.01 x10-4
1.45 x10-5
Cu
2+ c
on
cen
tra
tio
n (
pp
m)
PART IV: EXPERIMENTAL WORK
- 220 -
18.4.2 0.3% citric acid pH 2.8
Figure 18.4 and Figure 18.5 are typical examples of the real-time change in
projected HAp mineral mass content in response to exposure to 0.3% citric acid pH
2.8 solution with 22.5 ppm Cu2+
concentration in both increasing and decreasing
Cu2+
concentration sequences respectively.
Figure 18.4 shows that the HAp projected mineral mass content decreased
from 0.640 g/cm2 to 0.625 g/cm
2 in 20 h. This reduction represents only a 2.3% loss
in the projected HAp mineral mass over 20 h. While for Figure 18.5 the HAp
projected mineral mass content decreased from 0.600 g/cm2 to 0.580g/cm
2 in ≈20 h
which represents a 3.3% loss in the projected mineral content over ≈20 h.
PART IV: EXPERIMENTAL WORK
- 221 -
y=a+bxr2=0.784 FitStdErr=0.00241
a=0.640
b=-0.000731
0 5 10 15 20 25
Time (h)
0.62
0.625
0.63
0.635
0.64
0.645
0.65
Min
era
l m
ass (
g/c
m2)
FIGURE 18.4 Typical example of the change in projected HAp mineral mass content over
a period of ≈20 h in response to 0.3% citric acid pH 2.8 with 22.5 ppm Cu2+
demineralisation solution at increasing Cu2+
concentration sequence TABLE 18.4 Statistical analysis, for the data in Figure 18.4, using TableCurve 2D
®
Value SE t-value 95% Confidence Limits
a (g/cm2) 0.640 1.841e-04 3486.08 0.6394 0.6401
b (g/cm2/h) -7.31e-4 1.56e-05 -46.77 -7.62-4 - 7.00e-4
( Within 1 SD, 1 SD< < 2 SD, 2 SD< < 3 SD, 3 SD< < 4 SD)
PART IV: EXPERIMENTAL WORK
- 222 -
y=a+bxr2=0.895 FitStdErr=0.00224
a=0.600
b=-0.00103
0 5 10 15 20 25
Time (h)
0.575
0.58
0.585
0.59
0.595
0.6
0.605M
ine
ral m
ass (
g/c
m2)
FIGURE 18.5 Typical example of the change in projected HAp mineral mass content over
a period of ≈20 h in response to 0.3% citric acid pH 2.8 with 22.5 ppm Cu2+
demineralisation solution at decreasing Cu2+
concentration sequence
TABLE 18.5 Statistical analysis, for the data in Figure 18.4, using TableCurve 2D®
Value SE t-value 95% Confidence Limits
a (g/cm2`) 0.600 1.681e-04 3565.91 0.5993 0.6000
b (g/cm2/h) -1.03e-3 1.43e-05 -71.85 -1.06-3 - 9.99e-4
( Within 1 SD, 1 SD< < 2 SD, 2 SD< < 3 SD, 3 SD< < 4 SD)
PART IV: EXPERIMENTAL WORK
- 223 -
For each of the 28 demineralisation experiments using 0.3% acetic acid pH
2.8 with various Cu2+
concentrations, the projected mineral mass loss of each HAp
disc was continuously measured throughout the entire experimental duration. The
RDHAp and the SE were calculated and the results obtained are summarized in Table
18.6
TABLE 18.6 The RDHAp and SE for each demineralisation solution at different
Cu2+
concentrations at both increasing and decreasing concentration sequences.
0.3% citric acid pH 2.8
RDHAp (g/cm2/h) increasing Cu
2+
concentration sequence
RDHAp (g/cm2/h) decreasing Cu
2+
concentration sequence
HAp
disc1
SE
HAp
disc2
SE
HAp
disc1
SE
HAp
disc2
SE
180
5.95 x10-4 2.01x10-5 4.92 x10-4 1.43x10-5
6.31 x10-4 1.78x10-5 6.33 x10-4 1.67x10-5
150
6.42 x10-4 1.75x10-5 5.91 x10-4 1.57x10-5 6.36 x10-4 2.32x10-5 6.95 x10-4 3.21 x10-5
90
6.65 x10-4 1.43x10-5 6.12 x10-4 1.82x10-5 8.53 x10-4 2.13x10-5 7.97 x10-4 1.42 x10-5
45
7.06 x10-4 1.65x10-5 6.31 x10-4 1.18x10-5 9.42 x10-4 1.78x10-5 9.42 x10-4 1.30 x10-5
22.5
7.07 x10-4 1.42x10-5 7.31 x10-4 1.56x10-5 1.03 x10-3 1.43x10-5 1.07 x10-3 2.13 x10-5
11.25 8.78 x10-4 1.30x10-5 8.14 x10-4 2.70x10-5 1.16 x10-3 1.85x10-5 1.14 x10-3
1.47 x10-5
0 9.35 x10-4 1.56x10-5 8.22 x10-4 2.73x10-5 1.21 x10-3 1.45x10-5 1.20 x10-3
2.41 x10-5
Cu
2+ c
on
cen
tra
tio
n (
pp
m)
PART IV: EXPERIMENTAL WORK
- 224 -
18.5 Discussion
The results from this study highlighted the importance of the direct and sole
effect of Cu2+
as divalent metal cation on the kinetics of HAp dissolution, in
isolation from its antibacterial effect. The experiment investigated Cu2+
at a range of
concentrations from 0-180 ppm. Similar Cu2+
concentrations were used in other
previous studies (Afseth et al., 1984a, Brookes et al., 2003, Abdullah et al., 2006).
For caries-like conditions; Figure 18.2 and Figure 18.3 represent typical
examples of the change in projected HAp mineral mass content, over a period of ≈20
h when exposed to 0.1% acetic acid pH 4.0 with 22.5 ppm Cu2+
demineralisation
solution in increasing and decreasing concentration sequences respectively. In Figure
18.2 the change in mineral mass content (g/cm2) was plotted as a function of time
(h). The data showed a linear regression trend between the projected HAp mineral
mass content over time. The systematic gaps in the recording the data over the
experimental duration are because of more than one SMR cell been scanned
simultaneously over the experimental duration. In Figure 18.2, 606 data counts were
counted at a centrally located point on the permeable HAp disc over ≈20 h out of
which 27 data counts were outside 2 SD (4.5%).
Figure 18.3 represents the demineralisation in caries-like conditions similar
to those in Figure 18.2 but with the sequence of Cu2+
concentrations reversed. It
shows a similar linear regression trend in projected HAp mineral mass content over
the experimental duration. Six hundred and six data counts were collected at a
centrally located point on the permeable HAp disc over ≈20 h out of which 29 data
counts were outside 2 SD (4.7%).
Table 18.3 shows the calculated demineralisation rate and the SE for each of
the 28 experiments with various Cu2+
concentrations.
PART IV: EXPERIMENTAL WORK
- 225 -
The mean effect of Cu2+
on RDHAp at increasing concentration sequence
showed that as Cu2+
concentration increased over the range from 0-180 ppm, RDHAp
decreased (Figure 18.6). The reduction in RDHAp was statistically significant
(P≤0.05) for all Cu2+
concentrations investigated when compared to the control
group (0 ppm). These results are in accordance with the observations of Hein et al.
who reported caries reduction in hamsters with 50 ppm Cu2+
as copper sulphate in
drinking solutions (Hein, 1953). It also goes in accordance with the results obtained
by Afseth et al. who reported a significant reduction in caries in rats at 65 ppm Cu2+
applied as topical application (Afseth et al., 1984a). When the sequence of Cu2+
concentrations was reversed, as Cu2+
concentration decreased at a range of 180-0
ppm, RDHAp increased (Figure 18.7).
PART IV: EXPERIMENTAL WORK
- 226 -
0.00E+00
5.00E-05
1.00E-04
1.50E-04
2.00E-04
2.50E-04
3.00E-04
3.50E-04
4.00E-04
0 20 40 60 80 100 120 140 160 180 200
Copper concentration (ppm)
Dem
inera
lisati
on
rate
(g
/cm
2/h
)
mean increasing sequence
FIGURE 18.6 The effect of Cu2+
at a range of 0 - 180 ppm on mean RDHAp at increasing
Cu2+
concentration sequence under caries-like conditions
FIGURE 18.7 The effect of Cu
2+ at a range of 180 - 0 ppm on mean RDHAp at decreasing
Cu2+
concentration sequence under caries-like conditions
0.00E+00
5.00E-05
1.00E-04
1.50E-04
2.00E-04
2.50E-04
3.00E-04
3.50E-04
4.00E-04
0 20 40 60 80 100 120 140 160 180 200
Copper concentration (ppm)
Dem
inera
lisati
on
rate
(g
/cm
2/h
)
mean decreasing sequence
PART IV: EXPERIMENTAL WORK
- 227 -
The results of this study also show that the differences in reduction of RDHAp
obtained with 150 and 180 ppm Cu2+
concentration were not statistically significant
when compared to the reduction observed with 90 ppm Cu2+
. These results are in
agreement with those published by Brookes et al.(2003); as illustrated in Figure
18.8.
Figure 18.8 (a) The effect of Cu2+
concentration on phosphate released from powdered
enamel published by Brookes et al.(2003) after the conversion of mmol/L to ppm; (b)
Example of the effect of Cu2+
at a range of 0-180 ppm on mean RDHAp as observed in this
study
0.0
0.2
0.4
0.6
0.8
1.0
0 20 40 60 80 100 120 140 160 180 200
Cu2+ concentration (ppm)
Ph
osp
hate
lo
ss f
rom
en
am
el
po
wd
er
(a) (b)
0.0
0.2
0.4
0.6
0.8
1.0
0 20 40 60 80 100 120 140 160 180 200
Cu2+ concentration (ppm)H
Ap
dem
inera
lisati
on
rate
PART IV: EXPERIMENTAL WORK
- 228 -
0.00E+00
5.00E-05
1.00E-04
1.50E-04
2.00E-04
2.50E-04
3.00E-04
3.50E-04
4.00E-04
0 11.25 22.5 45 90 150 180
Copper concentration (ppm)
Dem
inera
lisati
on
rate
(g
/cm
2/h
)
mean increasing sequence mean decreasing sequence
3.52 x 10-4
3.11 x 10-4
2.46 x 10-4 2.47 x 10-4
1.65x 10-4
2.04 x 10-4
1.33 x 10-4
1.84 x 10-4
1.19 x 10-4
1.62 x 10-4
1.03 x 10-4
1.44 x 10-4
8.43 x 10-5
8.28 x 10-5
The average of each duplicate experiment, at each Cu2+
concentration, for
increasing and decreasing concentration sequence was calculated and presented in
Figure 18.9.
FIGURE 18.9 The effect of 0.1% acetic acid pH 4.0 with different Cu2+
concentrations
(ppm) on RDHAp (g/cm2/h) at both increasing and decreasing concentrations sequences
Figure 18.9 shows that Cu2+
had an inhibitory effect on the RDHAp. The
percentage reduction in RDHAp detected in this experiment was around 75%
reduction in caries-like conditions at Cu2+
concentration of 180 ppm. This percentage
is less than the percentage reduction in caries detection reported by Rosalen et al.
(1996a) who reported 82% reduction at 150 ppm Cu2+
concentration. The difference
in reduction rate detected can be attributed to the principle difference between the
two studies. The Rosalen et al. (1996a) study was an in vivo study, while this study
is in vitro. In addition, the effect of Cu2+
concentration in this study is determined
from its direct effect on HAp reflected as a change in RDHAp whereas in other studies
the effect of Cu2+
is measured indirectly by its effect on caries score.
PART IV: EXPERIMENTAL WORK
- 229 -
For erosion-like conditions Figure 18.4 and Figure 18.5 are typical examples
of the change in projected HAp mineral mass content, over a period of ≈20 h when
exposed to 0.3% citric acid pH 2.8 demineralisation solution containing 22.5 ppm
Cu2+
at increasing and decreasing concentration sequences respectively. The change
in mineral mass content (g/cm2) was plotted as a function of time (h). The data
showed a linear regression trend between the projected HAp mineral mass content
over time. The systematic gaps in recording the data over the experimental duration
were because of more than one SMR cell been scanned simultaneously over the
experimental duration. In Figure 18.4, 606 data counts were counted at a centrally
located point on the permeable HAp disc over ≈20 h out of which 31 data counts
were outside 2 SD (5.1%).
Figure 18.5 represents the demineralisation in erosion-like conditions similar
to those in Figure 18.4 but with the sequence reversed. It shows a similar linear
regression trend in projected HAp mineral mass content over the experimental
duration. Six hundred and six data counts were collected at a centrally located point
on the permeable HAp disc over ≈20 h out of which 30 data counts were outside 2
SD (4.9%).
Table 18.6 shows the calculated demineralisation rate and the SE for each of
the 28 experiments with various Cu2+
concentrations.
The mean effect of Cu2+
on RDHAp at increasing concentration sequences
showed that as Cu2+
concentration increased at a range of 0 - 180 ppm, the RDHAp
decreased (Figure 18.10). The reduction in RDHAp was statistically significant (P ≤
0.05) for all Cu2+
concentrations investigated when compared to the control group (0
ppm). However, when the sequence of Cu2+
concentrations was reversed, as Cu2+
PART IV: EXPERIMENTAL WORK
- 230 -
concentration decreased at a range of 180-0 ppm, the RDHAp increased (Figure
18.11).
FIGURE 18.10 The effect of Cu
2+ at a range of 0 - 180 ppm on mean RDHAp at increasing
Cu2+
concentration sequence under erosion-like conditions
FIGURE 18.11 The effect of Cu2+
at a range of 180 - 0 ppm on mean RDHAp at decreasing
Cu2+
concentration sequence under erosion-like conditions
4.0E-04
5.0E-04
6.0E-04
7.0E-04
8.0E-04
9.0E-04
1.0E-03
0 20 40 60 80 100 120 140 160 180 200
Copper concentration (ppm)
Dem
inera
lisati
on
rate
(g
/cm
2/h
)
mean increasing sequence
6.00E-04
7.00E-04
8.00E-04
9.00E-04
1.00E-03
1.10E-03
1.20E-03
1.30E-03
0 20 40 60 80 100 120 140 160 180 200Copper concentration (ppm)
Dem
inera
lisati
on
rate
(g
/cm
2/h
)
mean decreasing sequence
PART IV: EXPERIMENTAL WORK
- 231 -
FIGURE 18.12 The effect of 0.3% citric acid pH 2.8 with different Cu
2+ concentrations
(ppm) on RDHAp (g/cm2/h) at both increasing and decreasing concentrations sequences
The average of each duplicate experiment, at each Cu2+
concentration, at both
increasing and decreasing Cu2+
concentration sequences were calculated and shown
in Figure 18.12. The dose response data obtained from this study demonstrated a
significant and direct effect of Cu2+
on RDHAp from the minimal investigated
concentration of 11.25 ppm. However, Cu2+
concentrations of 150 ppm and 180 ppm
did not show a statistically significant reduction in RDHAp. These results are similar
to the results obtained from Brookes et al. (2003) (Figure 7.1).
Figure 18.12 shows that the mean RDHAp for Cu2+
decreasing sequence
experiments is higher than the RDHAp for Cu2+
increasing sequence experiments. The
justification remains unclear and requires further investigations.
All the series of 7 different Cu2+
concentrations, whether at increasing or
decreasing concentration sequence under conditions resembling artificial caries or
erosion, showed the same trend in RDHAp reduction/increase. The reversibility in
0.0E+00
2.0E-04
4.0E-04
6.0E-04
8.0E-04
1.0E-03
1.2E-03
1.4E-03
0 11.25 22.5 45 90 150 180
Copper concentration (ppm)
Dem
inera
lisati
on
rate
(g
/cm
2/h
)
mean increasing sequence mean decreasing sequence
8.79 x 10-4
8.22 x 10-4
6.89 x 10-4
6.39 x 10-4
6.17 x 10-4
5.44 x 10-4
7.50 x 10-4
6.26 x 10-4 6.66 x 10-4
8.25 x 10-4
9.42 x 10-4
1.05 x 10-3
1.15 x 10-3
1.21 x 10-3
PART IV: EXPERIMENTAL WORK
- 232 -
RDHAp through the increased or decreased Cu2+
concentration sequence supports the
hypothesis that Cu2+
under the experimental conditions does not permanently change
the HAp solid phase. Instead it affected the calcium-rich layer (stern layer) or
adhered to the HAp surface blocking the dissolution pit (Wang et al., 2005).
18.6 Conclusions
In conclusion, the results of this study showed the direct inhibitory effect of
Cu2+
as the divalent metal cation on HAp dissolution kinetics from the minimal
investigated concentration as 11.25 ppm. The reversibility of the effect suggests a
surface controlled action rather than change in the bulk composition. It demonstrates
the potential usefulness of Cu2+
as a preventive agent against caries and erosion.
- 233 -
PART V: GENERAL DISCUSSION, CONCLUSIONS,
CLINICAL IMPLICATIONS AND RECOMMENDED
FUTURE WORKS
PART V: GENERAL DISCUSSION, CONCLUSIONS, CLINICAL IMPLICATION AND
RECOMMENDED FUTURE WORKS
- 234 -
CHAPTER 19
General Discussion, Conclusions, Clinical Implications
and Future Works
19.1 General discussion
In order to develop an effective preventive strategy for mineral loss in dental
caries and erosion, it is essential to fully understand the physico-chemical processes
involved in these two conditions and the factors affecting them. Unfortunately many
aspects of the dental enamel demineralisation processes are still poorly understood.
For example, the direct effects of various divalent cations-enamel interactions,
relevant to demineralisation need further investigations and deeper understanding.
Therefore, the main aim of this thesis was to investigate the effect of Zn2+
, Sr2+
and
Cu2+
, as divalent metal cations, on HAp dissolution kinetics relevant to dental caries
and erosion-like conditions.
Ideally dental enamel should have been used. However, it was decided to use
permeable compressed sintered HAp discs instead of dental enamel due to the
uniformity and homogeneity of its structure compared to enamel. HAp has been
extensively used in research as a model system for dental enamel (Margolis and
Moreno, 1985, Anderson, 1993, Elliott et al., 2005).
PART V: GENERAL DISCUSSION, CONCLUSIONS, CLINICAL IMPLICATION AND
RECOMMENDED FUTURE WORKS
- 235 -
Most previous studies on Zn2+
and Cu2+
were aiming at investigating their
antimicrobial effect. However the scope of interest of this thesis was to investigate
the direct and sole effect of divalent cations on the kinetics of HAp dissolution.
As part of this study, new methodologies have been devised. This included
modification and optimisation of the SMR technique to obtain sufficient and
statistically reliable data over short period of 24 h or less (Chapter 10). Further the
developments of the research protocol which involved multiple experiments to
investigate the effect of changing various experimental parameters on HAp
dissolution kinetics. These studies included the characterization of the different types
of HAp discs using XRD, XMT and SMR, the effect of various demineralisation
solutions with range of pH on the RDHAp, the effect of demineralisation solution
circulation speed on RDHAp and the effect of high Sr2+
concentrations on HAp
dissolution kinetics. These studies are described in Chapters 11-15.
Studying the effect of divalent cations on the HAp dissolution kinetics via
exposing a single HAp disc to a series of demineralisation solutions containing
certain cations concentrations in both increasing and decreasing concentration
sequence for 20 h at each concentration separated by 30 min of washing by de-
ionised water, has proved to be a successful approach in evaluating the
persistence/lack of persistence of the effect of the divalent cation under investigation.
This experimental approach provided an insight to the different mechanisms through
which the various divalent cations under investigation affected the HAp dissolution
kinetics.
The results obtained from the effect of Sr2+
on RDHAp (Chapter 17) showed
that as Sr2+
concentrations were increased the RDHAp decreased, and when the Sr2+
PART V: GENERAL DISCUSSION, CONCLUSIONS, CLINICAL IMPLICATION AND
RECOMMENDED FUTURE WORKS
- 236 -
concentrations were subsequently decreased, the RDHAp continued to decrease. This
“persistence” of Sr2+
effect on HAp dissolution was demonstrated in its ability to
decrease RDHAp whether it was investigated at an increasing or decreasing
concentration sequence. These results support the hypothesis that Sr2+
substitutes
some Ca2+
in the HAp forming Sr-Ca-phosphates phase. The results of this
substitution should lead to the formation of a less stable phase (Sr-Ca-phosphates)
(LeGeros, 1991, Grynpas, 1993, Kikuchi et al., 1994) due to the difference in size
between Sr2+
and Ca2+
ions (Section6.4). However the explanation for the reduction
in RDHAp that was observed from the results of this study, can be justified by the
critically low Sr2+
concentrations investigated (0–30 ppm) which lead to less than
10% strontium substituted apatites. This comes in agreement with the results
reported by (Li et al., 2007) and (Verbeeck et al., 1981).
The results shown in Chapter 16 on the effect of using a range of Zn2+
concentrations (0-20 ppm) on RDHAp demonstrated that Zn2+
incorporated into caries
and erosion-like demineralisation conditions, provided an inhibitory effect. As Zn2+
concentrations were increased the RDHAp decreased, but when the Zn2+
concentrations were subsequently decreased, the RDHAp increased again. This lack of
“persistence” of Zn2+
effect on HAp disc dissolution suggests that Zn2+
exerts its
effect through an adsorption mechanism (Stötzel et al., 2009), rather than
incorporation into the crystal lattice mechanism as suggested in earlier studies
(Mayer et al., 1994, Li et al., 2008, Ren et al., 2009).
Cu2+
showed a similar effect as Zn2+
, suggesting similarly a surface
controlled effect rather than long term effect in reducing RDHAp under dental caries
and erosion-like conditions. However, the metallic taste and ability to cause teeth
PART V: GENERAL DISCUSSION, CONCLUSIONS, CLINICAL IMPLICATION AND
RECOMMENDED FUTURE WORKS
- 237 -
discolorations of Cu2+
will be limitations of its incorporation into therapeutic agents
aiming at the prevention of dental caries and erosion.
Comparison between the results of the effects of the three divalent metal
cations at 20 ppm concentration shows that Sr2+
provides the best protection against
HAp dissolution under both caries and erosion like conditions (58% and 50%
respectively). Copper demonstrates a slightly lower inhibitory effect (53% and 15%
reduction in RDHAp under caries and erosion like conditions respectively). Zinc
demonstrated the lowest efficacy with 38% reduction in RDHAp under caries like
conditions and 41% reduction in RDHAp under erosion like conditions. However,
although as discussed in sections 16.6, 17.6 and 18.6, the mechanisms are different
for the different ions, the dissolution inhibitions are similar.
Strontium and copper showed more protection for HAp against dissolution
when exposed to acetic acid pH 4.0, while zinc was more protective under the
erosive like conditions of citric acid pH 2.8. The exact reason behind this finding is
not known and more research is needed in this area. However it is an interesting
finding to be taken in consideration while selecting a suitable divalent cation when
designing a therapeutic regimen, or to be incorporated as a food and drink modifier
to protect against dental caries or erosion.
19.2 Conclusions
In this thesis, the effect of three divalent cations; Zn2+
, Sr2+
and Cu2+
, on the
physical-chemistry influencing HAp dissolution kinetics, under simulated cariogenic
and erosive conditions relevant to the oral environment was studied using an SMR
technique.
PART V: GENERAL DISCUSSION, CONCLUSIONS, CLINICAL IMPLICATION AND
RECOMMENDED FUTURE WORKS
- 238 -
The following conclusions were drawn:
1. SMR has been shown to be a highly suitable technique for investigating the
effect of cations on the kinetics of HAp dissolution. Among its advantages are
its accuracy in obtaining real-time quantitative measurements, the way it
allows alteration of the experimental conditions if required, to simulate the
more dynamic environment mimicking the oral cavity, without interrupting the
experiment.
2. SMR has previously been successfully used in experiments investigating
de/remineralisation over long period of time extending up to several weeks;
however the results in this thesis demonstrated that the SMR technique is also
capable of obtaining quantitatively reliable data with high accuracy and
precision over short time of 24 h or less.
3. The use of Zn2+
, Sr2+
and Cu2+
as therapeutic agents should not be simply
confined to their role as antiplaque and calculus agents, or for the treatment
and prevention of tooth hypersensitivity. Instead the ions’ use should be
expanded to include prevention of dental caries and erosion by directly
inhibiting dental tissue dissolution.
4. It was observed that Zn2+
and Cu2+
decreased RDHAp through a surface
controlled mechanism whereas Sr2+
decreased RDHAp through a solid phase
change. This information will be useful as part of the development of
therapeutic products which include these ions for the prevention of dental
caries and erosion.
PART V: GENERAL DISCUSSION, CONCLUSIONS, CLINICAL IMPLICATION AND
RECOMMENDED FUTURE WORKS
- 239 -
19.3 Clinical implications
Dental caries and erosion are worldwide problems, affecting populations in
both industrial and developing countries. According to WHO 2003 (Petersen, 2003)
dental caries alone affected approximately five billion people worldwide, and
prevalence of dental erosion has increased in recent years. The recent increase in
dental erosion might be due to a real increase in the disease due to faulty oral
hygiene habits and/or diet with high erosive potential, or due to the increased
awareness of the disease by both dentists and patients. Dental caries and erosion
form a real problem and their control is a challenge.
In the past it was thought that dental caries and erosion are irreversible
progressive dental tissue diseases. Nowadays with more research in the field, it has
been realized that enamel and dentine constantly undergo through alternating
demineralisation and remineralisation according to the surrounding oral
environment. It is also known that demineralisation can be stopped at early stages of
its development and remineralisation of very early lesions is possible. This depends
on the early detection and proper management of the condition via therapeutic agents
capable of controlling demineralisation and facilitating remineralisation of the
affected enamel.
Nowadays the concept of minimally invasive dentistry is more appreciated by
both dentists as well as by patients (Wilson, 2007). It is based on three basic
principles; prevention, less intrusive treatment, and conservation of healthy tissues.
The research interest in discovering and developing therapeutic agents that inhibit
demineralisation and ideally facilitate remineralisation of dental enamel has
increased recently. Historically, the ion of most interest in prevention of enamel
demineralisation was fluoride. The discovery of fluoride caries-reducing effect was a
PART V: GENERAL DISCUSSION, CONCLUSIONS, CLINICAL IMPLICATION AND
RECOMMENDED FUTURE WORKS
- 240 -
landmark in the history of dentistry. Until now almost all successful preventive
treatments contain fluoride. Fluoride cariostatic effectiveness does not only lay in its
effect on the apatite crystal lattice but also in its inhibition of mineral dissolution,
inhibition of acid formation by dental plaque bacteria and promotion of
remineralisation. Another element of interest to recent research is silver in its
divalent and trivalent cation forms. Silver as a trivalent metal cation has
demonstrated its ability in preventing against dental caries through its bacteriostatic
effect. Most recent researches on salivary proteins have demonstrated that statherin
and a subunit of protein STN21 have considerable effect in preventing HAp
demineralisation, and these peptides can be used as therapeutic agents for the
prevention or treatment of erosive and carious demineralisation.
In this thesis the three divalent cations of interest (Zn2+
, Sr2+
and Cu2+
)
showed positive results in their anti-carious and anti-erosive effect with promising
clinical implications.
19.3.1 Zinc
Zn2+
has been incorporated in oral hygiene products. It has been used in
toothpastes and mouthwashes for its antiplaque effect and for its capability to reduce
oral malodor. This is accomplished through its ability to alter bacterial metabolic
activity leading to reduction in bacterial growth and capability to adhere to tooth
surfaces. However, the results of this study have demonstrated that Zn2+
has a direct
effect on HAp dissolution kinetics under caries and erosion-like conditions. This
effect is significant even at low concentrations such as 5 ppm Zn2+
. This expands the
potential usefulness of Zn2+
in playing a role as a therapeutic agent added to
toothpastes and mouthwashes aiming at caries and erosion prevention. However, the
results of this thesis have demonstrated that the surface effect of Zn2+
in inhibiting
PART V: GENERAL DISCUSSION, CONCLUSIONS, CLINICAL IMPLICATION AND
RECOMMENDED FUTURE WORKS
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HAp dissolution should be taken into account in the design of the new Zn2+
containing therapeutic agents, for example allowing long term release of Zn2+
or
more frequent applications this can be achieved through the incorporation of Zn2+
into chewing gum or mouthwashes. Zn2+
toxicity should not be of concern (Section
5.1) as it does not have to be added in high concentrations to provide the preventive
effect.
19.3.2 Strontium
The second divalent metal cation studied in this thesis was Sr2+
. One of the
main clinical uses of Sr2+
is for the management of osteoporosis. Sr2+
stimulates
osteoblast cell activities and inhibits osteoclast cell differentiation, reducing in bone
resorption. This characteristic of Sr2+
has also led to its being favoured in dental
implants, by introducing Sr2+
as component in some bioactive glass materials to
facilitate the integration between the dental implant and bone.
Sr2+
has been also used for the prevention and management of tooth
hypersensitivity. Strontium chloride has been introduced commercially as the first
tubular occluding agent in Sensodyne™ Original toothpaste (Dowell and Addy,
1983). Sensodyne™ Rapid Relief is one of the latest products on the market to
manage tooth hypersensitivity with strontium acetate as a key ingredient. In order for
the Sr2+
to effectively block the dentinal tubules and reduce tooth sensitivity it has to
be incorporated at high concentration (80,000 ppm of strontium acetate) (Layer and
Hughes, 2010).
The results presented in this thesis have demonstrated a direct anti-carious
and anti-erosive effect of Sr2+
through its incorporation into the apatite lattice
forming strontium calcium phosphate which lowers the HAp dissolution rate when
applied in low concentration (0-30 ppm). A potential clinical implication arising
PART V: GENERAL DISCUSSION, CONCLUSIONS, CLINICAL IMPLICATION AND
RECOMMENDED FUTURE WORKS
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from this study is the manufacture of toothpastes or mouthwashes with these low
Sr2+
concentrations for caries and erosion prevention.
Another clinical implication is the use of Sr2+
in dental cements and glass
ionomer cements. It will not only have the advantage of being more radiopaque
which allow better follow up for caries progression, but the Sr2+
containing glass
ionomer cement will also have the advantage of providing a local anti-carious and
anti-erosive effect.
19.3.3 Copper
While Cu2+
has been used for its antimicrobial effect against dental plaque
bacteria causing caries and periodontal diseases, not much attention has been given
to the direct effect of Cu2+
in reducing the RDHAp. Cu2+
and Zn2+
both share the same
mechanism of affecting the kinetics of HAp dissolution. However the salty metallic
taste of Cu2+
and tooth discolouration might be a major drawback to its use in
therapeutic agents for the prevention of dental caries and erosion.
19.4 Recommended future works
1) Studies of dental enamel: In this thesis, the SMR technique was successfully
used to demonstrate the inhibitory effect of the three investigated divalent cations
(Zn2+
, Sr2+
and Cu2+
) on RDHAp. However, knowing that dental enamel consists
mainly of impure form of HAp, which contains multiple impurities, it would be
beneficial for the results of this study to be used as a base for a future work that
involves applying the same experiments using dental enamel.
2) Lower concentrations of investigated cations: The results of this thesis have
shown that Zn2+
, Sr2+
and Cu2+
, significantly reduced RDHAp even at the minimal
PART V: GENERAL DISCUSSION, CONCLUSIONS, CLINICAL IMPLICATION AND
RECOMMENDED FUTURE WORKS
- 243 -
investigated concentrations. However no concentrations less than 5 ppm were
investigated. It would be of interest in future works to investigate the same cations at
lower concentrations in an attempt to determine the lowest significantly effective
dose for each of the three cations.
3) The use of other techniques: As scanning microradiography is a powerful
technique concerned with quantifying changes in projected mineral mass content
over a period of time it would be interesting in a future study to combine the SMR
technique with another technique such as scanning electron microscopy (SEM) or
atomic force microscopy (AFM). The scanning electron microscope can be used to
reveal information about the sample including external morphology (texture),
chemical composition, and crystalline structure and orientation of materials making
up the sample. Using the energy dispersive X-ray spectroscopy (EDS) mode, SEM
will be useful in qualitatively or semi-quantitatively determining the chemical
compositions at selected point locations on the sample. Therefore, SMR and SEM
could complement each other in a future work to obtain more detailed information
about the mechanisms through which the investigated cations affect RDHap. Through
applying both techniques we might be able to get a better understanding of whether
the divalent cations inhibit the RDHAp through adhering to the surface blocking
dissolution nuclei or through replacing calcium ions within the apatite lattice altering
the physico-chemical properties of the apatite.
4) Testing of therapeutic agents: Dental caries and erosion are still considered a
significant problem affecting societies in both industrial as well as developing
countries. Every effort should be made to control these diseases, whether by
prevention or treatment. The world of dentistry is moving more towards non-
invasive dentistry and the industrial companies are more along the lines of producing
PART V: GENERAL DISCUSSION, CONCLUSIONS, CLINICAL IMPLICATION AND
RECOMMENDED FUTURE WORKS
- 244 -
preventive agents such as toothpastes, mouthwashes, gel etc. Therefore, considerably
more work can be done applying the SMR technology on studying different
therapeutic agents when their efficacy and effect on demineralisation/
remineralisation need to be tested. The SMR technique benefits from accuracy and
high precision in real-time detection of minute changes in mineral mass content, as
well as allowing for the possibility of altering experimental conditions without
interrupting the experiment. Taking these advantages into consideration, the SMR
technique has superiority over other available techniques of mineral quantification.
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APPENDIX I: ABSTRACTS FOR CONFERENCE PRESENTATIONS AND PAPERS IN
PREPARATION
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APPENDIX I
ABSTRACTS FOR CONFERENCE PRESENTATIONS
AND PAPERS IN PREPARATION
APPENDIX I: ABSTRACTS FOR CONFERENCE PRESENTATIONS AND PAPERS IN
PREPARATION
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List of conferences presentations that have arisen from the work
presented in this thesis
1. H. Lingawi, M.E. Barbour, P. Anderson
Effect of Replenishment Rate of Demineralisation Solutions on Hydroxyapatite
Dissolution Kinetics Studied Using Scanning Microradiography, International Caries
Research Conference, Montpellier, France (July, 2010)
ORAL PRESENTATION
2. H. Lingawi, M.E. Barbour, R.J.M. Lynch, P. Anderson
Effect of Zinc ions (Zn2+
) on Hydroxyapatite Dissolution Kinetics Studied Using
Scanning Microradiography, 2nd
UK Zinc meeting, London, UK, (October, 2010)
ORAL PRESENTATION
3. H. Lingawi, M.E. Barbour, P. Anderson
Effect of Demineralisation Solutions Circulation Rates on Hydroxyapatite
Dissolution Kinetics Studied Using Scanning Microradiography, William Harvey
Day, QMUL, (October, 2010)
POSTER PRESENTATION
4. H. Lingawi, M.E. Barbour, R.J.M. Lynch, P. Anderson
Effect of Zinc (Zn2+
) and Strontium (Sr2+
) Ions on Hydroxyapatite Thermodynamic
Dissolution Kinetics, Weybridge Scientific Conference, Surry, UK, (April 2011)
ORAL PRESENTATION
5. H. Lingawi, M.E. Barbour, R.J.M. Lynch, P. Anderson
Effect of Zinc (Zn2+
) and Strontium (Sr2+
) Ions on Hydroxyapatite Dissolution
Relevant to Dental Caries and Erosion, International Association of Paediatric
Dentistry, Athens, Greece, (June 2011)
POSTER PRESENTATION
APPENDIX I: ABSTRACTS FOR CONFERENCE PRESENTATIONS AND PAPERS IN
PREPARATION
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6. H. Lingawi, M.E. Barbour, R.J.M. Lynch, P. Anderson
Effect of Zinc as Divalent Metal Cation on Hydroxyapatite Dissolution Kinetics
Studied Using Scanning Microradiography, International Caries Research
Conference, Kaunas, Lithuania (July, 2011)
ORAL PRESENTATION
7. H. Lingawi, M.E. Barbour, P. Anderson
Cariostatic Influence of Sr2+
on Hydroxyapatite-disc Tooth Analogue
Demineralisation, The British Society of Oral and Dental Research, Sheffield, UK
(September 2011)
ORAL PRESENTATION
8. H. Lingawi, M.E. Barbour, P. Anderson
Effect of Sr2+
on Hydroxyapatite Dissolution Kinetics Studied Using Scanning
Microradiography, William Harvey Day, QMUL, (October, 2011)
POSTER PRESENTATION
9. H. Lingawi, M.E. Barbour, P. Anderson
Effect of Divalent Metal Cations on Hydroxyapatite Dissolution Relevant to Dental
Caries and Erosion, London Oral Biology Club, QMUL, London (November, 2011)
ORAL PRESENTATION
APPENDIX I: ABSTRACTS FOR CONFERENCE PRESENTATIONS AND PAPERS IN
PREPARATION
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List of papers in preparation that have arisen from the work
presented in this thesis
1. H. Lingawi, P. Anderson
Real-time Scanning Microradiography for the Quantitative Measurements of
Dissolution Kinetics of Compressed Hydroxyapatite Pellets
Scanning
2. H. Lingawi, M.E. Barbour, R.J.M. Lynch, P. Anderson
Effect of Zinc (Zn2+
) and Strontium (Sr2+
) Ions on Hydroxyapatite Dissolution
Relevant to Dental Caries and Erosion
Caries Research
APPENDIX I: ABSTRACTS FOR CONFERENCE PRESENTATIONS AND PAPERS IN
PREPARATION
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Published abstracts for oral presentations
1. H. Lingawi, M.E. Barbour, P. Anderson. Effect of Replenishment Rate of
Demineralisation Solutions on Hydroxyapatite Dissolution Kinetics Studied
Using Scanning Microradiography. The European Organization for Caries
Research Conference, France, (July, 2010)
Abstract
The replenishment of demineralising solution adjacent to a dissolving surface has
considerable influence on the rate of dissolution of solids. This is particularly
pertinent to dissolution studies of enamel, and similar studies of model systems for
dental caries using compressed powders of hydroxyapatite as the substrate. As part
of an overall investigation of the fundamental mechanisms influencing kinetics of
enamel and hydroxyapatite dissolution, the aim was to compare the dissolution rates
of compressed hydroxyapatite (HAP) powder discs as a function of replenishment
rate of demineralising solution, using scanning microradiography (SMR).
Compressed HAP powder discs product of Plasma –Biotal with 20 wt% nominal
porosity were sterilised, coated with acid-resistant varnish on all surfaces except one,
preconditioned, and located in an SMR cell volume 1.96 cm3. Demineralising
solution (0.1% acetic acid buffered with 1M KOH, pH 4.0) was pumped at various
replenishments rates using a variable speed circulating pump. The rate of HAP
dissolution (RDHAP) was measured using SMR at a single centrally located point on
each disc for periods of 24 h at 22°C. Each measurement was repeated in triplicate.
The mean RDHAP was; 6.58x10-6
, 1.18 x10-4
, 1.70 x10-4
, 2.40 x10-4
, 2.72 x10-4
,
3.13 x10-4
, 3.16 x10-4
g.cm-2.h-1 at circulation speeds of 0, 0.19, 0.39, 0.58, 0.80,
0.97 and 1.17cm3.min-1 respectively.
The RDHAP statistically significantly increased for circulation speeds up to 0.78
cm3.min-1, but did not change significantly at higher speeds.
This study demonstrates that the solution composition in contact with a
demineralising HAP surface achieved by sufficient replenishment rate, or stirring, is
an important parameter in HAP dissolution studies. Diffusive transport of dissolved
substrate away from the dissolving HAP surface will influence the kinetics of the
process.
APPENDIX I: ABSTRACTS FOR CONFERENCE PRESENTATIONS AND PAPERS IN
PREPARATION
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2. H. Lingawi, M.E. Barbour, R.J.M. Lynch, P. Anderson. Effect of Zinc Ions
(Zn2+
) on Hydroxyapatite Dissolution Kinetics Studied Using Scanning
Microradiography. The European Organization for Caries Research
Conference, Lithuania, (July, 2011)
Abstract
Zinc (Zn2+
) is a dietary essential trace element necessary for various body functions.
It is used in toothpaste for its anti-calculus properties and reducing oral malodour,
but it may also have a role in inhibiting dissolution kinetics of enamel’s principal
inorganic component; hydroxyapatite (HAp).
The aim of this study was to investigate the effect of Zn2+
on surface physical
chemistry influencing HAp dissolution by measuring the rate of HAp dissolution
(RDHAp) under strictly controlled thermodynamic conditions relevant to caries and
erosion using scanning microradiography (SMR) at a range of Zn2+
concentrations.
Compressed sintered HAp discs (Plasma-Biotal, UK) were coated with acid-resistant
varnish on all surfaces except one, and located in an SMR cell. A bulk solution of
0.1% acetic acid pH4, divided into five (1 litre bottle) with the addition of 0, 5, 10,
15, 20 ppm Zn2+
respectively was prepared. 0.3% citric acid pH2.8 solutions were
similarly prepared.
The demineralising solution was circulated at 0.80cm3/min, and the RDHAp was
measured using SMR at a single centrally located point on each disc for 24h at 22°C.
Each experiment was repeated in duplicate for both increasing, and decreasing, Zn2+
concentrations.
For acetic acid, the mean RDHAp decreased significantly (p< 0.05) from 4.38 x10-4
(with no Zn2+
added) to 3.81x10-4
, 3.19x10-4
, 3.02x10-4
, and 2.71x10-4
g/cm2/h at
Zn2+
concentrations of 5, 10, 15 and 20 ppm respectively.
For citric acid, the mean RDHAp decreased significantly (p<0.05) from 3.12 x10-3
(with no Zn2+
added) to 2.83x10-3
, 2.73x10-3
, 2.45x10-3
and 1.83x10-3
g/cm2/h at Zn
2+
concentrations of 5, 10, 15 and 20 ppm respectively.
This study demonstrates that Zn2+
decreased RDHAp under strictly controlled
thermodynamic conditions relevant to caries and erosion, possibly due to inhibition
of dissolution nuclei on the HAp surfaces.
APPENDIX I: ABSTRACTS FOR CONFERENCE PRESENTATIONS AND PAPERS IN
PREPARATION
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3. H. Lingawi, M.E. Barbour, P. Anderson. Cariostatic Influence of Sr2+
on
Hydroxyapatite-Disc Tooth Analogue Demineralisation. The British Society
of Oral and Dental Research, Sheffield, UK (September 2011).
Abstract
Objectives: Strontium (Sr2+
) has been demonstrated to be cariostatic. The evidence is
controversial and the exact mechanism by which strontium decreases dental caries is
unclear. Our aim is to study the effect of the divalent metal cation Sr2+
on the
kinetics of porous hydroxyapatite (HAp) disc dissolution using scanning
microradiography (SMR) under artificial caries and erosion conditions.
Methods: Compressed 1mm thick sintered HAp discs (Plasma-Biotal, UK. 20wt%
nominal porosity) used as tooth analogues, were preconditioned, coated with acid-
resistant varnish on all surfaces leaving one surface exposed, and located centrally in
SMR cell. 1L 0.1% acetic acid pH 4.0 (caries conditions) and 0.3% citric acid pH 2.8
(erosion conditions) demineralising solutions were prepared with each of 0, 5, 10, 20
and 30 ppm Sr2+
respectively. Demineralising solution was circulated at 0.80
cm3/min, and the HAp demineralisation rate (RDHAp) was measured at a single
centrally located point on each disc for 24 h at 22±1°C using SMR. Each experiment
was repeated twice for both increasing, and decreasing sequences of Sr2+
concentrations.
Results: Caries conditions: mean RDHAp decreased significantly from 3.40x10-4
(0
ppm Sr2+
) to 2.73x10-4
(5 ppm), 1.88x10-4
(10 ppm), 1.44x10-4
(20 ppm), and
1.15x10-4
(30 ppm) g/cm2/h for increasing concentration sequence, and from 1.47
x10-4
(30ppm Sr2+
) to 1.24x10-4
(20 ppm), 1.04x10-4
(10 ppm), 6.10x10-5
(5 ppm)
and 2.39x10-4
(0 ppm) g/cm2/h for decreasing concentration sequence.
Erosion conditions: mean RDHAp decreased significantly from 4.22 x10-3
(0 ppm
Sr2+
) to 4.02x10-3
(5 ppm), 3.58x10-3
(10 ppm), 3.45x10-3
(20 ppm) and 2.83x10-3
(30 ppm) g/cm2/h for increasing concentration sequence, and from 3.94x10
-3 (30
ppm Sr2+
) to 3.55x10-3
(20 ppm), 3.19x10-3
(10 ppm) , 2.58x10-3
(5 ppm) , and
3.65x10-3
(0 ppm) g/cm2/h for decreasing concentration sequence.
Conclusion: Sr2+
decreased RDHAp under strictly controlled thermodynamic
conditions relevant to dental caries and erosion. The non-reversibility in RDHAp
throughout the increasing and decreasing Sr2+
sequences may be due to lasting
effects of phase changes in HAp. This study demonstrates the potential usefulness of
Sr2+
in caries prevention.
APPENDIX I: ABSTRACTS FOR CONFERENCE PRESENTATIONS AND PAPERS IN
PREPARATION
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4. H. Lingawi, M.E. Barbour, P. Anderson. Effect of Sr2+
on Hydroxyapatite
Demineralisation Using Scanning Microradiography. The European
Organization for Caries Research Conference, Brazil, (June, 2012)
Abstract
The literature on the cariostatic effects of strontium (Sr2+
) remains controversial and
the mechanism is obscure. The aim was to study the effect of Sr2+ in the
demineralising solution on the kinetics of hydroxyapatite (HAp) dissolution using
scanning microradiography (SMR) under artificial caries and erosion conditions.
Hydroxyapatite discs (Plasma-Biotal, UK. 20wt% porosity) 1mm thick sintered,
were used as enamel analogues, coated with acid-resistant varnish leaving one
surface exposed, and located in an SMR cell. Demineralising solutions of 0.1%
acetic acid pH4 simulating caries conditions, and 0.3% citric acid pH2.8, simulating
erosive conditions were circulated through the SMR cells. The rate of
demineralisation of the HAp discs (RDHAp) was measured using SMR. Further SMR
measurements were carried out using identical demineralising conditions, but with
increasing Sr2+
concentrations of 5, 10, 20 and 30 ppm, and SMR measurements
were continued for each case. The SMR measurements were then repeated at
decreasing Sr2+
concentrations (30, 20, 10, 5 and 0 ppm).
Results for Caries-like conditions showed RDHAp decreased (3.40x10-4
, 2.73x10-4
,
1.88x10-4
1.44x10-4
, 1.15x10-4
g.cm-2
.h-1
) at increasing Sr2+
concentrations. RDHAp
also decreased (1.47x10-4
, 1.24x10-4
, 1.04x10-4
, 6.10x10-5
g.cm-2
.h-1
) at decreasing
Sr2+
concentrations, except for 2.39x10-4
g.cm-2
.h-1
at 0 ppm.
Erosive-like conditions RDHAp decreased (4.22x10-3
, 4.02x10-3
, 3.58x10-3
, 3.45x10-3
,
3.12x10-3
g.cm-2
.h-1
) at increasing Sr2+
concentrations. RDHAp also decreased
(3.94x10-3
, 3.55x10-3
, 3.19x10-3
, 2.58x10-3
g.cm-2
.h-1
) at decreasing Sr2+
concentrations except for 3.65x10-3
g.cm-2
.h-1
at 0 ppm.
In conclusion, Sr2+
decreased RDHAp under strictly controlled thermodynamic
conditions relevant to caries and erosion. However, this decrease was not reversed
when the Sr2+
concentration was subsequently decreased. This pattern of the
influence of Sr2+
may result from the partial inclusion of Sr2+
into the HAp lattice.
APPENDIX I: ABSTRACTS FOR CONFERENCE PRESENTATIONS AND PAPERS IN
PREPARATION
- 265 -
Samples of poster presentations
1. H. Lingawi, M.E. Barbour, P. Anderson. Effect of Demineralisation Solutions
Circulation Rate on Hydroxyapatite Dissolution Kinetics Studied Using
Scanning Microradiography. William Harvey Day, QMUL (October, 2010)
APPENDIX I: ABSTRACTS FOR CONFERENCE PRESENTATIONS AND PAPERS IN
PREPARATION
- 266 -
2. H. Lingawi, M.E. Barbour, P. Anderson. Effect of Strontium Ions on
Hydroxyapatite Dissolution Kinetics Studied Using Scanning
Microradiography. William Harvey Day, QMUL, (October, 2011).
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